RNA containing composition for treatment of tumor diseases

ABSTRACT

The present invention relates to RNA containing compositions for use in the treatment or prophylaxis of tumor and/or cancer diseases, to a pharmaceutical composition, to a kit and to uses of the RNA containing compositions for the treatment or prophylaxis of tumor and/or cancer diseases.

This application claims the priority of European Application No. 15001191.4, filed Apr. 22, 2015, the entirety of which is incorporated herein by reference.

The sequence listing that is contained in the file named “CRVCP0175US.txt”, which is 18,426 KB (as measured in Microsoft Windows) and was created on Apr. 22, 2016, is filed herewith by electronic submission and is incorporated herein by reference.

INTRODUCTION

The present invention relates to RNA containing compositions for use in the treatment or prophylaxis of tumor and/or cancer diseases, to a pharmaceutical composition, to a kit and to uses of the RNA containing compositions for the treatment or prophylaxis of tumor and/or cancer diseases.

Cancer, also known as malignant tumor, describes a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. In 2012, about 14.1 million new cases of cancer occurred globally (not including skin cancer other than melanoma).

The standard treatments of cancer include chemotherapy, radiation and surgery, wherein these treatments are applied individually or in combination. Other treatments apply cancer immunotherapy which is focused on stimulating the immune system through vaccination or adoptive cellular immunotherapy to elicit an anti-tumor response.

Some approaches use gene therapy and genetic vaccination for treatment of cancer or other tumor diseases. Gene therapy and genetic vaccination are molecular medicine methods which are based on the introduction of nucleic acids into cells or into tissues of a patient. Subsequently the information coded by the nucleic acids introduced is processed in the organism, i.e. resulting in expression of a therapeutic peptide or protein or expression of an antigen which is coded by the nucleic acids.

Conventional gene therapeutic methods, including gene therapy and genetic vaccination are based on the use of DNA molecules in order to transfer the desired genetic information into the cell. Various methods have been developed for introducing DNA into cells, such as calcium phosphate transfection, polybrene transfection, protoplast fusion, electroporation, microinjection and lipofection. DNA viruses may likewise be used as a DNA vehicle achieving a very high transfection rate. The use of DNA entails the risk of the DNA being inserted into an intact gene of the host cell's genome by e.g. recombination. In this case the affected gene may be mutated and inactivated or may give rise to misinformation. Another risk of using DNA as a pharmaceutical agent is the risk of inducing pathogenic anti-drug antibodies (anti-DNA antibodies) in the patient, which may result in a (possibly fatal) immune response.

The use of RNA as a gene therapeutic agent or genetic vaccine is substantially safer, because RNA does not involve the risk of being integrated into the genome inducing an undesired pathogenic induction of anti-drug antibodies.

Thus RNA expression systems have considerable advantages over DNA expression systems in gene therapy and in genetic vaccination although it is known in the prior art or rather assumed for a long time that the instability of mRNA or of RNA in general may be problem in the application of medical methods based on RNA expression systems.

The instability of RNA is in particular due to RNA-degrading enzymes (ribonucleases-RNases). There are also many further processes which destabilize RNA, wherein interaction between the RNA and proteins often appears to play a crucial role. Some measures for increasing the stability of RNA have been proposed, so enabling the use thereof as a gene therapy agent or RNA vaccine.

For solving the problem of ex vivo RNA stability the European patent application EP 1 083 232 A1 describes a method for introducing RNA, in particular mRNA, into cells and organisms, in which the RNA forms a complex with a cationic peptide or protein.

The application of mRNA is known for the treatment and/or prophylaxis of cancer. For example the international patent application WO 03/051401 A2 describes a pharmaceutical composition comprising at least one mRNA, which contains at least one region that codes for an antigen from a tumor, combined with an aqueous solvent and preferably with a cytokine e.g. GM-CSF. The pharmaceutical composition is proposed to be used for therapy and/or prophylaxis against cancer.

The international patent application WO 2006/008154 A1 discloses an mRNA mixture for vaccinating against tumor diseases, wherein at least one type of mRNA contains at least one tumor antigen-coding region. At least one other mRNA contains at least one type of an immunogenic protein-coding region.

Nevertheless there is still a need for an effective treatment of tumor diseases and especially for the treatment of cancer. Therefore it is the object of the underlying invention to provide an approach for effective treatment of tumor diseases wherein tumor tissue and cancer cells are specifically destroyed.

This object is solved by the subject matter of the claims. Particularly, the object underlying the present invention is solved according to a first aspect by an RNA containing composition for use in the treatment or prophylaxis of tumor and/or cancer diseases. According to further aspects of the invention the object is solved by a pharmaceutical composition, by a kit or kit of parts, and by a method of treatment of tumor or cancer diseases.

DEFINITIONS

For the sake of clarity and readability the following scientific background information and definitions are provided. Any technical features disclosed thereby can be part of each and every embodiment of the invention. Additional definitions and explanations can be provided in the context of this disclosure.

Immune system: The immune system may protect organisms from infection. If a pathogen breaks through a physical barrier of an organism and enters this organism, the innate immune system provides an immediate, but non-specific response. If pathogens evade this innate response, vertebrates possess a second layer of protection, the adaptive immune system. Here, the immune system adapts its response during an infection to improve its recognition of the pathogen. This improved response is then retained after the pathogen has been eliminated, in the form of an immunological memory, and allows the adaptive immune system to mount faster and stronger attacks each time this pathogen is encountered. According to this, the immune system comprises the innate and the adaptive immune system. Each of these two parts contains so called humoral and cellular components.

Immune response: An immune response may typically either be a specific reaction of the adaptive immune system to a particular antigen (so called specific or adaptive immune response) or an unspecific reaction of the innate immune system (so called unspecific or innate immune response).

Adaptive immune system: The adaptive immune system is composed of highly specialized, systemic cells and processes that eliminate or prevent pathogenic growth. The adaptive immune response provides the vertebrate immune system with the ability to recognize and remember specific pathogens (to generate immunity), and to mount stronger attacks each time the pathogen is encountered. The system is highly adaptable because of somatic hypermutation (a process of increased frequency of somatic mutations), and V(D)J recombination (an irreversible genetic recombination of antigen receptor gene segments). This mechanism allows a small number of genes to generate a vast number of different antigen receptors, which are then uniquely expressed on each individual lymphocyte. Because the gene rearrangement leads to an irreversible change in the DNA of each cell, all of the progeny (offspring) of that cell will then inherit genes encoding the same receptor specificity, including the Memory B cells and Memory T cells that are the keys to long-lived specific immunity. Immune network theory is a theory of how the adaptive immune system works, that is based on interactions between the variable regions of the receptors of T cells, B cells and of molecules made by T cells and B cells that have variable regions.

Adaptive immune response: The adaptive immune response is typically understood to be antigen-specific. Antigen specificity allows for the generation of responses that are tailored to specific antigens, pathogens or pathogen-infected cells. The ability to mount these tailored responses is maintained in the body by “memory cells”. Should a pathogen infect the body more than once, these specific memory cells are used to quickly eliminate it. In this context, the first step of an adaptive immune response is the activation of naïve antigen-specific T cells or different immune cells able to induce an antigen-specific immune response by antigen-presenting cells. This occurs in the lymphoid tissues and organs through which naïve T cells are constantly passing. Cell types that can serve as antigen-presenting cells are inter alia dendritic cells, macrophages, and B cells. Each of these cells has a distinct function in eliciting immune responses. Dendritic cells take up antigens by phagocytosis and macropinocytosis and are stimulated by contact with e.g. a foreign antigen to migrate to the local lymphoid tissue, where they differentiate into mature dendritic cells. Macrophages ingest particulate antigens such as bacteria and are induced by infectious agents or other appropriate stimuli to express MHC molecules. The unique ability of B cells to bind and internalize soluble protein antigens via their receptors may also be important to induce T cells. Presenting the antigen on MHC molecules leads to activation of T cells which induces their proliferation and differentiation into armed effector T cells. The most important function of effector T cells is the killing of infected cells by CD8+ cytotoxic T cells and the activation of macrophages by Th1 cells which together make up cell-mediated immunity, and the activation of B cells by both Th2 and Th1 cells to produce different classes of antibody, thus driving the humoral immune response. T cells recognize an antigen by their T cell receptors which do not recognize and bind antigen directly, but instead recognize short peptide fragments e.g. of pathogen-derived protein antigens, which are bound to MHC molecules on the surfaces of other cells.

Cellular immunity/cellular immune response: Cellular immunity relates typically to the activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to an antigen. In a more general way, cellular immunity is not related to antibodies but to the activation of cells of the immune system. A cellular immune response is characterized e.g. by activating antigen-specific cytotoxic T-lymphocytes that are able to induce apoptosis in body cells displaying epitopes of an antigen on their surface, such as virus-infected cells, cells with intracellular bacteria, and cancer cells displaying tumor antigens; activating macrophages and natural killer cells, enabling them to destroy pathogens; and stimulating cells to secrete a variety of cytokines that influence the function of other cells involved in adaptive immune responses and innate immune responses.

Humoral immunity/humoral immune response: Humoral immunity refers typically to antibody production and the accessory processes that may accompany it. A humoral immune response may be typically characterized, e.g., by Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation and memory cell generation. Humoral immunity also typically may refer to the effector functions of antibodies, which include pathogen and toxin neutralization, classical complement activation, and opsonin promotion of phagocytosis and pathogen elimination.

Innate immune system: The innate immune system, also known as non-specific immune system, comprises the cells and mechanisms that defend the host from infection by other organisms in a non-specific manner. This means that the cells of the innate system recognize and respond to pathogens in a generic way, but unlike the adaptive immune system, it does not confer long-lasting or protective immunity to the host. The innate immune system may be e.g. activated by ligands of pathogen-associated molecular patterns (PAMP) receptors, e.g. Toll-like receptors (TLRs) or other auxiliary substances such as lipopolysaccharides, TNF-alpha, CD40 ligand, or cytokines, monokines, lymphokines, interleukins or chemokines, immunostimulatory nucleic acids, immunostimulatory RNA (isRNA), CpG-DNA, antibacterial agents, or anti-viral agents. Typically a response of the innate immune system includes recruiting immune cells to sites of infection, through the production of chemical factors, including specialized chemical mediators, called cytokines; activation of the complement cascade; identification and removal of foreign substances present in organs, tissues, the blood and lymph, by specialized white blood cells; activation of the adaptive immune system through a process known as antigen presentation; and/or acting as a physical and chemical barrier to infectious agents.

Adjuvant/adjuvant component: An adjuvant or an adjuvant component in the broadest sense is typically a (e.g. pharmacological or immunological) agent or composition that may modify, e.g. enhance, the efficacy of other agents, such as a drug or vaccine. Conventionally the term refers in the context of the invention to a compound or composition that serves as a carrier or auxiliary substance for immunogens and/or other pharmaceutically active compounds. It is to be interpreted in a broad sense and refers to a broad spectrum of substances that are able to increase the immunogenicity of antigens incorporated into or co-administered with an adjuvant in question. In the context of the present invention an adjuvant will preferably enhance the specific immunogenic effect of the active agents of the present invention. Typically, “adjuvant” or “adjuvant component” has the same meaning and can be used mutually. Adjuvants may be divided, e.g., into immuno potentiators, antigenic delivery systems or even combinations thereof.

The term “adjuvant” is typically understood not to comprise agents which confer immunity by themselves. An adjuvant assists the immune system unspecifically to enhance the antigen-specific immune response by e.g. promoting presentation of an antigen to the immune system or induction of an unspecific innate immune response. Furthermore, an adjuvant may preferably e.g. modulate the antigen-specific immune response by e.g. shifting the dominating Th2-based antigen specific response to a more Th1-based antigen specific response or vice versa. Accordingly, an adjuvant may favourably modulate cytokine expression/secretion, antigen presentation, type of immune response etc.

Immunostimulatory/immunostimulating RNA: An immunostimulatory/immunostimulating RNA (isRNA) in the context of the invention may typically be a RNA that is able to induce an innate immune response itself. It usually does not have an open reading frame and thus does not provide a peptide-antigen or immunogen but elicits an innate immune response e.g. by binding to a specific kind of Toll-like-receptor (TLR) or other suitable receptors. Therefore immunostimulatory/immunostimulating RNAs are preferably non-coding RNAs. However, of course also mRNAs having an open reading frame and coding for a peptide/protein (e.g. an antigenic function) may induce an innate immune response.

Antigen: The term “antigen” refers typically to a substance which may be recognized by the immune system and may be capable of triggering an antigen-specific immune response, e.g. by formation of antibodies or antigen-specific T-cells as part of an adaptive immune response. An antigen may be a protein or peptide. In this context, the first step of an adaptive immune response is the activation of naïve antigen-specific T cells by antigen-presenting cells. This occurs in the lymphoid tissues and organs through which naïve T cells are constantly passing. The three cell types that can serve as antigen-presenting cells are dendritic cells, macrophages, and B cells. Each of these cells has a distinct function in eliciting immune responses. Tissue dendritic cells take up antigens by phagocytosis and macropinocytosis and are stimulated by infection to migrate to the local lymphoid tissue, where they differentiate into mature dendritic cells. Macrophages ingest particulate antigens such as bacteria and are induced by infectious agents to express MHC class II molecules. The unique ability of B cells to bind and internalize soluble protein antigens via their receptors may be important to induce T cells. By presenting the antigen on MHC molecules leads to activation of T cells which induces their proliferation and differentiation into armed effector T cells. The most important function of effector T cells is the killing of infected cells by CD8+ cytotoxic T cells and the activation of macrophages by Th1 cells which together make up cell-mediated immunity, and the activation of B cells by both Th2 and Th1 cells to produce different classes of antibody, thus driving the humoral immune response. T cells recognize an antigen by their T cell receptors which does not recognize and bind antigen directly, but instead recognize short peptide fragments e.g. of pathogens' protein antigens, which are bound to MHC molecules on the surfaces of other cells.

T cells fall into two major classes that have different effector functions. The two classes are distinguished by the expression of the cell-surface proteins CD4 and CD8. These two types of T cells differ in the class of MHC molecule that they recognize. There are two classes of MHC molecules—MHC class I and MHC class II molecules—which differ in their structure and expression pattern on tissues of the body. CD4+ T cells bind to a MHC class II molecule and CD8+ T cells to a MHC class I molecule. MHC class I and MHC class II molecules have distinct distributions among cells that reflect the different effector functions of the T cells that recognize them. MHC class I molecules present peptides of cytosolic and nuclear origin e.g. from pathogens, commonly viruses, to CD8+ T cells, which differentiate into cytotoxic T cells that are specialized to kill any cell that they specifically recognize. Almost all cells express MHC class I molecules, although the level of constitutive expression varies from one cell type to the next. But not only pathogenic peptides from viruses are presented by MHC class I molecules, also self-antigens like tumor antigens are presented by them. MHC class I molecules bind peptides from proteins degraded in the cytosol and transported in the endoplasmic reticulum. The CD8+ T cells that recognize MHC class I:peptide complexes at the surface of infected cells are specialized to kill any cells displaying foreign peptides and so rid the body of cells infected with viruses and other cytosolic pathogens. The main function of CD4+ T cells (CD4+ helper T cells) that recognize MHC class II molecules is to activate other effector cells of the immune system. Thus MHC class II molecules are normally found on B lymphocytes, dendritic cells, and macrophages, cells that participate in immune responses, but not on other tissue cells. Macrophages, for example, are activated to kill the intravesicular pathogens they harbour, and B cells to secrete immunoglobulins against foreign molecules. MHC class II molecules are prevented from binding to peptides in the endoplasmic reticulum and thus MHC class II molecules bind peptides from proteins which are degraded in endosomes. They can capture peptides from pathogens that have entered the vesicular system of macrophages, or from antigens internalized by immature dendritic cells or the immunoglobulin receptors of B cells. Pathogens that accumulate in large numbers inside macrophage and dendritic cell vesicles tend to stimulate the differentiation of Th1 cells, whereas extracellular antigens tend to stimulate the production of Th2 cells. Th1 cells activate the microbicidal properties of macrophages and induce B cells to make IgG antibodies that are very effective of opsonising extracellular pathogens for ingestion by phagocytic cells, whereas Th2 cells initiate the humoral response by activating naïve B cells to secrete IgM, and induce the production of weakly opsonising antibodies such as IgG1 and IgG3 (mouse) and IgG2 and IgG4 (human) as well as IgA and IgE (mouse and human).

Epitope (also called “antigen determinant”): T cell epitopes may comprise fragments preferably having a length of about 6 to about 20 or even more amino acids, e.g. fragments as processed and presented by MHC class I molecules, preferably having a length of about 8 to about 10 amino acids, e.g. 8, 9, or 10, (or even 11, or 12 amino acids), or fragments as processed and presented by MHC class II molecules, preferably having a length of about 13 or more amino acids, e.g. 13, 14, 15, 16, 17, 18, 19, 20 or even more amino acids, wherein these fragments may be selected from any part of the amino acid sequence. These fragments are typically recognized by T cells in form of a complex consisting of the peptide fragment and an MHC molecule. B cell epitopes are typically fragments located on the outer surface of (native) protein or peptide antigens.

Vaccine: A vaccine is typically understood to be a prophylactic or therapeutic material providing at least one antigen or antigenic function. The antigen or antigenic function may stimulate the body's adaptive immune system to provide an adaptive immune response.

Antigen-providing mRNA: An antigen-providing mRNA may typically be an mRNA, having at least one open reading frame that can be translated by a cell or an organism provided with that mRNA. The product of this translation is a peptide or protein that may act as an antigen, preferably as an immunogen. The product may also be a fusion protein composed of more than one immunogen, e.g. a fusion protein that consist of two or more epitopes, peptides or proteins, wherein the epitopes, peptides or proteins may be linked by linker sequences.

Bi-/multicistronic mRNA: An bi-/multicistronic mRNA typically may have two (bicistronic) or more (multicistronic) coding sequences (cds) (also often referred to as open reading frames (ORF)). A coding sequence/an open reading frame in this context is a sequence of several nucleotide triplets (codons) that can be translated into a peptide or protein. Translation of such an mRNA yields two (bicistronic) or more (multicistronic) distinct translation products (provided the coding sequences/ORFs are not identical). For expression in eukaryotes such mRNAs may for example comprise an internal ribosomal entry site (IRES) sequence.

5′-CAP-Structure: A 5′-CAP is typically a modified nucleotide (CAP analogue), particularly a guanine nucleotide, added to the 5′ end of an mRNA molecule. Preferably, the 5′-CAP is added using a 5′-5′-triphosphate linkage (also named m7GpppN). Further examples of 5′-CAP structures include glyceryl, inverted deoxy abasic residue (moiety), 4′,5′ methylene nucleotide, 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides, alpha-nucleotide, modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3′,4′-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety, 3′-3′-inverted abasic moiety, 3′-2′-inverted nucleotide moiety, 3′-2′-inverted abasic moiety, 1,4-butanediol phosphate, 3′-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3′-phosphate, 3′phosphorothioate, phosphorodithioate, or bridging or non-bridging methylphosphonate moiety. These modified 5′-CAP structures may be used in the context of the present invention to modify the mRNA sequence of the inventive composition. Further modified 5′-CAP structures which may be used in the context of the present invention are CAP1 (additional methylation of the ribose of the adjacent nucleotide of m7GpppN), CAP2 (additional methylation of the ribose of the 2^(nd) nucleotide downstream of the m7GpppN), CAP3 (additional methylation of the ribose of the 3^(rd) nucleotide downstream of the m7GpppN), CAP4 (additional methylation of the ribose of the 4^(th) nucleotide downstream of the m7GpppN), ARCA (anti-reverse CAP analogue), modified ARCA (e.g. phosphothioate modified ARCA), inosine, N1-methyl-guanosine, 2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

In the context of the present invention, a 5′ cap structure may also be formed in chemical RNA synthesis or RNA in vitro transcription (co-transcriptional capping) using cap analogues, or a cap structure may be formed in vitro using capping enzymes (e.g., commercially available capping kits)

Cap analogue: A cap analogue refers to a non-polymerizable di-nucleotide that has cap functionality in that it facilitates translation or localization, and/or prevents degradation of the RNA molecule when incorporated at the 5′ end of the RNA molecule. Non-polymerizable means that the cap analogue will be incorporated only at the 5′terminus because it does not have a 5′ triphosphate and therefore cannot be extended in the 3′ direction by a template-dependent RNA polymerase.

Cap analogues include, but are not limited to, a chemical structure selected from the group consisting of m7GpppG, m7GpppA, m7GpppC; unmethylated cap analogues (e.g., GpppG); dimethylated cap analogue (e.g., m2,7GpppG), trimethylated cap analogue (e.g., m2,2,7GpppG), dimethylated symmetrical cap analogues (e.g., m7Gpppm7G), or anti reverse cap analogues (e.g., ARCA; m7,2′OmeGpppG, m7,2′dGpppG, m7,3′OmeGpppG, m7,3′dGpppG and their tetraphosphate derivatives) (Stepinski et al., 2001. RNA 7(10):1486-95).

Further cap analogues have been described previously (U.S. Pat. No. 7,074,596, WO 2008/016473, WO 2008/157688, WO 2009/149253, WO 2011/015347, and WO 2013/059475). The synthesis of N⁷-(4-chlorophenoxyethyl) substituted dinucleotide cap analogues has been described recently (Kore et al. (2013) Bioorg. Med. Chem. 21(15): 4570-4).

Fragments of proteins: “Fragments” of proteins or peptides in the context of the present invention may, typically, comprise a sequence of a protein or peptide as defined herein, which is, with regard to its amino acid sequence (or its encoded nucleic acid molecule), N-terminally and/or C-terminally truncated compared to the amino acid sequence of the original (native) protein (or its encoded nucleic acid molecule). Such truncation may thus occur either on the amino acid level or correspondingly on the nucleic acid level. A sequence identity with respect to such a fragment as defined herein may therefore preferably refer to the entire protein or peptide as defined herein or to the entire (coding) nucleic acid molecule of such a protein or peptide. In the context of antigens such fragment may have a length of about 6 to about 20 or even more amino acids, e.g. fragments as processed and presented by MHC class I molecules, preferably having a length of about 8 to about 10 amino acids, e.g. 8, 9, or 10, (or even 6, 7, 11, or 12 amino acids), or fragments as processed and presented by MHC class II molecules, preferably having a length of about 13 or more amino acids, e.g. 13, 14, 15, 16, 17, 18, 19, 20 or even more amino acids, wherein these fragments may be selected from any part of the amino acid sequence. These fragments are typically recognized by T-cells in form of a complex consisting of the peptide fragment and an MHC molecule, i.e. the fragments are typically not recognized in their native form. Fragments of proteins or peptides (e.g. in the context of antigens) may comprise at least one epitope of those proteins or peptides. Furthermore also domains of a protein, like the extracellular domain, the intracellular domain or the transmembrane domain and shortened or truncated versions of a protein may be understood to comprise a fragment of a protein. Preferably, a fragment of a protein comprises a functional fragment of the protein, which means that the fragment exerts the same effect or functionality as the whole protein it is derived from.

Variants of proteins: “Variants” of proteins or peptides as defined in the context of the present invention may be generated, having an amino acid sequence which differs from the original sequence in one or more mutation(s), such as one or more substituted, inserted and/or deleted amino acid(s). Preferably, these fragments and/or variants have the same biological function or specific activity compared to the full-length native protein, e.g. its specific antigenic property. “Variants” of proteins or peptides as defined in the context of the present invention may comprise conservative amino acid substitution(s) compared to their native, i.e. non-mutated physiological, sequence. Those amino acid sequences as well as their encoding nucleotide sequences in particular fall under the term variants as defined herein. Substitutions in which amino acids, which originate from the same class, are exchanged for one another are called conservative substitutions. In particular, these are amino acids having aliphatic side chains, positively or negatively charged side chains, aromatic groups in the side chains or amino acids, the side chains of which can enter into hydrogen bridges, e.g. side chains which have a hydroxyl function. This means that e.g. an amino acid having a polar side chain is replaced by another amino acid having a likewise polar side chain, or, for example, an amino acid characterized by a hydrophobic side chain is substituted by another amino acid having a likewise hydrophobic side chain (e.g. serine (threonine) by threonine (serine) or leucine (isoleucine) by isoleucine (leucine)). Insertions and substitutions are possible, in particular, at those sequence positions which cause no modification to the three-dimensional structure or do not affect the binding region. Modifications to a three-dimensional structure by insertion(s) or deletion(s) can easily be determined e.g. using CD spectra (circular dichroism spectra) (Urry, 1985, Absorption, Circular Dichroism and ORD of Polypeptides, in: Modern Physical Methods in Biochemistry, Neuberger et al. (ed.), Elsevier, Amsterdam).

A “variant” of a protein or peptide may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity over a stretch of 10, 20, 30, 50, 75 or 100 amino acids of such protein or peptide. Furthermore, variants of proteins or peptides as defined herein, which may be encoded by a nucleic acid molecule, may also comprise those sequences, wherein nucleotides of the encoding nucleic acid sequence are exchanged according to the degeneration of the genetic code, without leading to an alteration of the respective amino acid sequence of the protein or peptide, i.e. the amino acid sequence or at least part thereof may not differ from the original sequence within the above meaning. Preferably, a variant of a protein comprises a functional variant of the protein, which means that the variant exerts the same effect or functionality as the protein it is derived from.

Identity of a sequence: In order to determine the percentage to which two sequences are identical, e.g. nucleic acid sequences or amino acid sequences as defined herein, preferably the amino acid sequences encoded by a nucleic acid sequence of the polymeric carrier as defined herein or the amino acid sequences themselves, the sequences can be aligned in order to be subsequently compared to one another. Therefore, e.g. a position of a first sequence may be compared with the corresponding position of the second sequence. If a position in the first sequence is occupied by the same component (residue) as is the case at a position in the second sequence, the two sequences are identical at this position. If this is not the case, the sequences differ at this position. If insertions occur in the second sequence in comparison to the first sequence, gaps can be inserted into the first sequence to allow a further alignment. If deletions occur in the second sequence in comparison to the first sequence, gaps can be inserted into the second sequence to allow a further alignment. The percentage to which two sequences are identical is then a function of the number of identical positions divided by the total number of positions including those positions which are only occupied in one sequence. The percentage to which two sequences are identical can be determined using a mathematical algorithm. A preferred, but not limiting, example of a mathematical algorithm which can be used is the algorithm of Karlin et al. (1993), PNAS USA, 90:5873-5877 or Altschul et al. (1997), Nucleic Acids Res., 25:3389-3402. Such an algorithm is integrated in the BLAST program. Sequences which are identical to the sequences of the present invention to a certain extent can be identified by this program.

Monocistronic mRNA: A monocistronic mRNA may typically be an mRNA, that comprises only one coding sequence (open reading frame). A coding sequence/open reading frame in this context is a sequence of several nucleotide triplets (codons) that can be translated into a peptide or protein.

Nucleic acid: The term nucleic acid means any DNA or RNA molecule and is used synonymous with polynucleotide. Wherever herein reference is made to a nucleic acid or nucleic acid sequence encoding a particular protein and/or peptide, said nucleic acid or nucleic acid sequence, respectively, preferably also comprises regulatory sequences allowing in a suitable host, e.g. a human being, its expression, i.e. transcription and/or translation of the nucleic acid sequence encoding the particular protein or peptide.

Peptide: A peptide is a polymer of amino acid monomers. Usually the monomers are linked by peptide bonds. The term “peptide” does not limit the length of the polymer chain of amino acids. In some embodiments of the present invention a peptide may for example contain less than 50 monomer units. Longer peptides are also called polypeptides, typically having 50 to 600 monomeric units, more specifically 50 to 300 monomeric units.

Pharmaceutically effective amount: A pharmaceutically effective amount in the context of the invention is typically understood to be an amount that is sufficient to induce an immune response or to trigger the desired therapeutical effect.

Protein: A protein typically consists of one or more peptides and/or polypeptides folded into 3-dimensional form, facilitating a biological function.

Poly(C) sequence: A poly(C) sequence is typically a long sequence of cytosine nucleotides, typically about 10 to about 200 cytosine nucleotides, preferably about 10 to about 100 cytosine nucleotides, more preferably about 10 to about 70 cytosine nucleotides or even more, preferably about 20 to about 50, or even about 20 to about 30 cytosine nucleotides. A poly(C) sequence may preferably be located 3′ of the coding region comprised by a nucleic acid.

Poly(A) tail: A poly(A) tail also called “3′-poly(A) tail” or “Poly(A) sequence” is typically a long homopolymeric sequence of adenosine nucleotides of up to about 400 adenosine nucleotides, e.g. from about 25 to about 400, preferably from about 50 to about 400, more preferably from about 50 to about 300, even more preferably from about 50 to about 250, most preferably from about 60 to about 250 adenosine nucleotides, added to the 3′ end of an mRNA. In the context of the present invention, the poly(A) tail of an mRNA is preferably derived from a DNA template by RNA in vitro transcription. Alternatively, the poly(A) sequence may also be obtained in vitro by common methods of chemical synthesis without being necessarily transcribed from a DNA-progenitor. Moreover, poly(A) sequences, or poly(A) tails may be generated by enzymatic polyadenylation of the RNA.

Stabilized nucleic acid: A stabilized nucleic acid, typically, exhibits a modification increasing resistance to in vivo degradation (e.g. degradation by an exo- or endo-nuclease) and/or ex vivo degradation (e.g. by the manufacturing process prior to vaccine administration, e.g. in the course of the preparation of the vaccine solution to be administered). Stabilization of RNA can, e.g., be achieved by providing a 5′-CAP-Structure, a poly(A) tail, or any other UTR-modification. It can also be achieved by backbone-modification or modification of the G/C-content or the C-content of the nucleic acid. Various other methods are known in the art and conceivable in the context of the invention.

Carrier/polymeric carrier: A carrier in the context of the invention may typically be a compound that facilitates transport and/or complexation of another compound. Said carrier may form a complex with said other compound. A polymeric carrier is a carrier that is formed of a polymer.

Cationic component: The term “cationic component” typically refers to a charged molecule, which is positively charged (cation) at a pH value of typically about 1 to 9, preferably of a pH value of or below 9 (e.g. 5 to 9), of or below 8 (e.g. 5 to 8), of or below 7 (e.g. 5 to 7), most preferably at physiological pH values, e.g. about 7.3 to 7.4. Accordingly, a cationic peptide, protein or polymer according to the present invention is positively charged under physiological conditions, particularly under physiological salt conditions of the cell in vivo. A cationic peptide or protein preferably contains a larger number of cationic amino acids, e.g. a larger number of Arg, His, Lys or Orn than other amino acid residues (in particular more cationic amino acids than anionic amino acid residues like Asp or Glu) or contains blocks predominantly formed by cationic amino acid residues. The definition “cationic” may also refer to “polycationic” components.

Vehicle: A vehicle is an agent, e.g. a carrier, that may typically be used within a pharmaceutical composition or vaccine for facilitating administering of the components of the pharmaceutical composition or vaccine to an individual. 3′-untranslated region (3′-UTR): A 3′-UTR is typically the part of an mRNA which is located between the protein coding region (i.e. the open reading frame) and the poly(A) sequence of the mRNA. A 3′-UTR of the mRNA is not translated into an amino acid sequence. The 3′-UTR sequence is generally encoded by the gene which is transcribed into the respective mRNA during the gene expression process. The genomic sequence is first transcribed into pre-mature mRNA, which comprises optional introns. The pre-mature mRNA is then further processed into mature mRNA in a maturation process. This maturation process comprises the steps of 5′-capping, splicing the pre-mature mRNA to excise optional introns and modifications of the 3′-end, such as polyadenylation of the 3′-end of the pre-mature mRNA and optional endo- or exonuclease cleavages etc. In the context of the present invention, a 3′-UTR corresponds to the sequence of a mature mRNA which is located 3′ to the stop codon of the protein coding region, preferably immediately 3′ to the stop codon of the protein coding region, and which extends to the 5′-side of the poly(A) sequence, preferably to the nucleotide immediately 5′ to the poly(A) sequence. The term “corresponds to” means that the 3′-UTR sequence may be an RNA sequence, such as in the mRNA sequence used for defining the 3′-UTR sequence, or a DNA sequence which corresponds to such RNA sequence. In the context of the present invention, the term “a 3′-UTR of a gene”, such as “a 3′-UTR of an albumin gene”, is the sequence which corresponds to the 3′-UTR of the mature mRNA derived from this gene, i.e. the mRNA obtained by transcription of the gene and maturation of the pre-mature mRNA. The term “3′-UTR of a gene” encompasses the DNA sequence and the RNA sequence of the 3′-UTR.

5′-untranslated region (5′-UTR): A 5′-UTR is typically understood to be a particular section of messenger RNA (mRNA). It is located 5′ of the open reading frame of the mRNA. Typically, the 5′-UTR starts with the transcriptional start site and ends one nucleotide before the start codon of the open reading frame. The 5′-UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, for example, ribosomal binding sites or a 5′-Terminal Oligopyrimidine Tract. The 5′-UTR may be posttranscriptionally modified, for example by addition of a 5′-CAP. In the context of the present invention, a 5′UTR corresponds to the sequence of a mature mRNA which is located between the 5′-CAP and the start codon. Preferably, the 5′-UTR corresponds to the sequence which extends from a nucleotide located 3′ to the 5′-CAP, preferably from the nucleotide located immediately 3′ to the 5′-CAP, to a nucleotide located 5′ to the start codon of the protein coding region, preferably to the nucleotide located immediately 5′ to the start codon of the protein coding region. The nucleotide located immediately 3′ to the 5′-CAP of a mature mRNA typically corresponds to the transcriptional start site. The term “corresponds to” means that the 5′-UTR sequence may be an RNA sequence, such as in the mRNA sequence used for defining the 5′-UTR sequence, or a DNA sequence which corresponds to such RNA sequence. In the context of the present invention, the term “a 5′-UTR of a gene”, such as “a 5′-UTR of a TOP gene”, is the sequence which corresponds to the 5′-UTR of the mature mRNA derived from this gene, i.e. the mRNA obtained by transcription of the gene and maturation of the pre-mature mRNA. The term “5′-UTR of a gene” encompasses the DNA sequence and the RNA sequence of the 5′-UTR.

5′ Terminal Oligopyrimidine Tract (TOP): The 5′ terminal oligopyrimidine tract (TOP) is typically a stretch of pyrimidine nucleotides located at the 5′ terminal region of a nucleic acid molecule, such as the 5′ terminal region of certain mRNA molecules or the 5′ terminal region of a functional entity, e.g. the transcribed region, of certain genes. The sequence starts with a cytidine, which usually corresponds to the transcriptional start site, and is followed by a stretch of usually about 3 to 30 pyrimidine nucleotides. For example, the TOP may comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or even more nucleotides. The pyrimidine stretch and thus the 5′ TOP ends one nucleotide 5′ to the first purine nucleotide located downstream of the TOP. mRNA that contains a 5′ terminal oligopyrimidine tract is often referred to as TOP mRNA. Accordingly, genes that provide such messenger RNAs are referred to as TOP genes. TOP sequences have, for example, been found in genes and mRNAs encoding peptide elongation factors and ribosomal proteins.

TOP motif: In the context of the present invention, a TOP motif is a nucleic acid sequence which corresponds to a 5′ TOP as defined above. Thus, a TOP motif in the context of the present invention is preferably a stretch of pyrimidine nucleotides having a length of 3-30 nucleotides. Preferably, the TOP-motif consists of at least 3 pyrimidine nucleotides, preferably at least 4 pyrimidine nucleotides, preferably at least 5 pyrimidine nucleotides, more preferably at least 6 nucleotides, more preferably at least 7 nucleotides, most preferably at least 8 pyrimidine nucleotides, wherein the stretch of pyrimidine nucleotides preferably starts at its 5′ end with a cytosine nucleotide. In TOP genes and TOP mRNAs, the TOP-motif preferably starts at its 5′ end with the transcriptional start site and ends one nucleotide 5′ to the first purine residue in said gene or mRNA. A TOP motif in the sense of the present invention is preferably located at the 5′end of a sequence which represents a 5′-UTR or at the 5′ end of a sequence which codes for a 5′-UTR. Thus, preferably, a stretch of 3 or more pyrimidine nucleotides is called “TOP motif” in the sense of the present invention if this stretch is located at the 5′ end of a respective sequence, such as the inventive mRNA, the 5′-UTR element of the inventive mRNA, or the nucleic acid sequence which is derived from the 5′-UTR of a TOP gene as described herein. In other words, a stretch of 3 or more pyrimidine nucleotides which is not located at the 5′-end of a 5′-UTR or a 5′-UTR element but anywhere within a 5′-UTR or a 5′-UTR element is preferably not referred to as “TOP motif”.

TOP gene: TOP genes are typically characterised by the presence of a 5′ terminal oligopyrimidine tract. Furthermore, most TOP genes are characterized by a growth-associated translational regulation. However, also TOP genes with a tissue specific translational regulation are known. As defined above, the 5′-UTR of a TOP gene corresponds to the sequence of a 5′-UTR of a mature mRNA derived from a TOP gene, which preferably extends from the nucleotide located 3′ to the 5′-CAP to the nucleotide located 5′ to the start codon. A 5′-UTR of a TOP gene typically does not comprise any start codons, preferably no upstream AUGs (uAUGs) or upstream open reading frames (uORFs). Therein, upstream AUGs and upstream open reading frames are typically understood to be AUGs and open reading frames that occur 5′ of the start codon (AUG) of the open reading frame that should be translated. The 5′-UTRs of TOP genes are generally rather short. The lengths of 5′-UTRs of TOP genes may vary between 20 nucleotides up to 500 nucleotides, and are typically less than about 200 nucleotides, preferably less than about 150 nucleotides, more preferably less than about 100 nucleotides. Exemplary 5′-UTRs of TOP genes in the sense of the present invention are the nucleic acid sequences extending from the nucleotide at position 5 to the nucleotide located immediately 5′ to the start codon (e.g. the ATG) in the sequences according to SEQ ID Nos. 1-1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of the international patent application WO2013/143700 or homologs or variants thereof, whose disclosure is incorporated herewith by reference. In this context a particularly preferred fragment of a 5′UTR of a TOP gene is a 5′-UTR of a TOP gene lacking the 5′ TOP motif. The term ‘5′UTR of a TOP gene’ preferably refers to the 5′-UTR of a naturally occurring TOP gene.

Chemical synthesis of RNA: Chemical synthesis of relatively short fragments of oligonucleotides with defined chemical structure provides a rapid and inexpensive access to custom-made oligonucleotides of any desired sequence. Whereas enzymes synthesize DNA and RNA only in the 5′ to 3′ direction, chemical oligonucleotide synthesis does not have this limitation, although it is most often carried out in the opposite, i.e. the 3′ to 5′ direction. Currently, the process is implemented as solid-phase synthesis using the phosphoramidite method and phosphoramidite building blocks derived from protected nucleosides (A, C, G, and U), or chemically modified nucleosides.

To obtain the desired oligonucleotide, the building blocks are sequentially coupled to the growing oligonucleotide chain on a solid phase in the order required by the sequence of the product in a fully automated process. Upon the completion of the chain assembly, the product is released from the solid phase to the solution, deprotected, and collected. The occurrence of side reactions sets practical limits for the length of synthetic oligonucleotides (up to about 200 nucleotide residues), because the number of errors increases with the length of the oligonucleotide being synthesized. Products are often isolated by HPLC to obtain the desired oligonucleotides in high purity.

Chemically synthesized oligonucleotides find a variety of applications in molecular biology and medicine. They are most commonly used as antisense oligonucleotides, small interfering RNA, primers for DNA sequencing and amplification, probes for detecting complementary DNA or RNA via molecular hybridization, tools for the targeted introduction of mutations and restriction sites, and for the synthesis of artificial genes.

RNA In vitro transcription: The terms “RNA in vitro transcription” or “in vitro transcription” relate to a process wherein RNA is synthesized in a cell-free system (in vitro). DNA, particularly plasmid DNA, is used as template for the generation of RNA transcripts. RNA may be obtained by DNA-dependent in vitro transcription of an appropriate DNA template, which according to the present invention is preferably a linearized plasmid DNA template. The promoter for controlling in vitro transcription can be any promoter for any DNA-dependent RNA polymerase. Particular examples of DNA-dependent RNA polymerases are the T7, T3, and SP6 RNA polymerases. A DNA template for in vitro RNA transcription may be obtained by cloning of a nucleic acid, in particular cDNA corresponding to the respective RNA to be in vitro transcribed, and introducing it into an appropriate vector for in vitro transcription, for example into plasmid DNA. In a preferred embodiment of the present invention the DNA template is linearized with a suitable restriction enzyme, before it is transcribed in vitro. The cDNA may be obtained by reverse transcription of mRNA or chemical synthesis. Moreover, the DNA template for in vitro RNA synthesis may also be obtained by gene synthesis.

Methods for in vitro transcription are known in the art (see, e.g., Geall et al. (2013) Semin. Immunol. 25(2): 152-159; Brunelle et al. (2013) Methods Enzymol. 530:101-14). Reagents used in said method typically include:

1) a linearized DNA template with a promoter sequence that has a high binding affinity for its respective RNA polymerase such as bacteriophage-encoded RNA polymerases;

2) ribonucleoside triphosphates (NTPs) for the four bases (adenine, cytosine, guanine and uracil);

3) optionally a cap analogue as defined above (e.g. m7G(5′)ppp(5′)G (m7G));

4) a DNA-dependent RNA polymerase capable of binding to the promoter sequence within the linearized DNA template (e.g. T7, T3 or SP6 RNA polymerase);

5) optionally a ribonuclease (RNase) inhibitor to inactivate any contaminating RNase;

6) optionally a pyrophosphatase to degrade pyrophosphate, which may inhibit transcription;

7) MgCl₂, which supplies Mg²⁺ ions as a co-factor for the polymerase;

8) a buffer to maintain a suitable pH value, which can also contain antioxidants (e.g. DTT), and/or polyamines such as spermidine at optimal concentrations.

RNA, mRNA: RNA is the usual abbreviation for ribonucleic acid. It is a nucleic acid molecule, i.e. a polymer consisting of nucleotide monomers. These nucleotides are usually adenosine monophosphate (AMP), uridine monophosphate (UMP), guanosine monophosphate (GMP) and cytidine monophosphate (CMP) monomers or analogues thereof, which are connected to each other along a so-called backbone. The backbone is formed by phosphodiester bonds between the sugar, i.e. ribose, of a first and a phosphate moiety of a second, adjacent monomer. The specific order of the monomers, i.e. the order of the bases linked to the sugar/phosphate-backbone, is called the RNA sequence. Usually RNA may be obtainable by transcription of a DNA sequence, e.g., inside a cell. In eukaryotic cells, transcription is typically performed inside the nucleus or the mitochondria. In vivo, transcription of DNA usually results in the so-called premature RNA (also called pre-mRNA, precursor mRNA or heterogeneous nuclear RNA) which has to be processed into so-called messenger RNA, usually abbreviated as mRNA. Processing of the premature RNA, e.g. in eukaryotic organisms, comprises a variety of different posttranscriptional modifications such as splicing, 5′-capping, polyadenylation, export from the nucleus or the mitochondria and the like. The sum of these processes is also called maturation of RNA. The mature messenger RNA usually provides the nucleotide sequence that may be translated into an amino acid sequence of a particular peptide or protein. Typically, a mature mRNA comprises a 5′-cap, optionally a 5′UTR, an open reading frame, optionally a 3′UTR and a poly(A) tail.

In addition to messenger RNA, several non-coding types of RNA exist which may be involved in regulation of transcription and/or translation, and immunostimulation. Within the present invention the term “RNA” further encompasses any type of single stranded (ssRNA) or double stranded RNA (d5RNA) molecule known in the art, such as viral RNA, retroviral RNA and replicon RNA, small interfering RNA (siRNA), antisense RNA (asRNA), circular RNA (circRNA), ribozymes, aptamers, riboswitches, immunostimulating/immunostimulatory RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), and Piwi-interacting RNA (piRNA).

Fragment of a nucleic acid sequence, particularly an RNA: A fragment of a nucleic acid sequence consists of a continuous stretch of nucleotides corresponding to a continuous stretch of nucleotides in the full-length nucleic acid sequence which is the basis for the nucleic acid sequence of the fragment, which represents at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90% of the full-length nucleic acid sequence. Such a fragment, in the sense of the present invention, is preferably a functional fragment of the full-length nucleic acid sequence.

Variant of a nucleic acid sequence, particularly anRNA: A variant of a nucleic acid sequence refers to a variant of nucleic acid sequences which forms the basis of a nucleic acid sequence. For example, a variant nucleic acid sequence may exhibit one or more nucleotide deletions, insertions, additions and/or substitutions compared to the nucleic acid sequence from which the variant is derived. Preferably, a variant of a nucleic acid sequence is at least 40%, preferably at least 50%, more preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% identical to the nucleic acid sequence the variant is derived from. Preferably, the variant is a functional variant. A “variant” of a nucleic acid sequence may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% nucleotide identity over a stretch of 10, 20, 30, 50, 75 or 100 nucleotide of such nucleic acid sequence.

Intratumoral administration/application: The term “intratumoral administration/application” refers to the direct delivery of a pharmaceutical composition into or adjacent to a tumor or cancer and/or immediate vicinity of a tumor or cancer. Multiple injections into separate regions of the tumor or cancer are also included. Furthermore, intratumoral administration/application includes delivery of a pharmaceutical composition into one or more metastases.

Methods for intratumoral delivery of drugs are known in the art (Brincker, 1993. Crit. Rev. Oncol. Hematol. 15(2):91-8; Celikoglu et al., 2008. Cancer Therapy 6, 545-552). For example, the pharmaceutical composition can be administered by conventional needle injection, needle-free jet injection or electroporation or combinations thereof into the tumor or cancer tissue. The pharmaceutical composition can be injected directly into the tumor or cancer (tissue) with great precision using computer tomograpy, ultrasound, gamma camera imaging, positron emission tomography, or magnetic resonance tumor imaging. Further procedures are selected from the group including, but not limited to, direct intratumoral injection by endoscopy, bronchoscopy, cystoscopy, colonoscopy, laparoscope and catheterization.

Decoy receptors: Decoy receptors recognize certain growth factors or cytokines with high affinity and specificity, but are structurally incapable of signaling or presenting the agonist to signaling receptor complexes. They act as a molecular trap for the agonist and for signaling receptor components. A decoy receptor, or sink receptor, is a receptor that binds a ligand, inhibiting it from binding to its normal receptor. For instance, the receptor VEGFR-1 can prevent vascular endothelial growth factor (VEGF) from binding to the VEGFR-2.

Dominant negative receptors: Dominant negative receptors are variants of the particular receptor comprising dominant-negative (DN) mutations as leading to mutant polypeptides that disrupt the activity of the wild-type receptor when overexpressed.

DETAILED DESCRIPTION OF THE INVENTION

The RNA containing composition according to the invention comprises at least one RNA and is particularly provided for use in the treatment or prophylaxis of tumor and/or cancer diseases, wherein the RNA containing composition is preferably applied/administered intratumorally. It is especially preferred that the RNA containing composition is injected directly into tumor tissue. Alternatively, it is especially preferred that the RNA containing composition is injected adjacent to or in close proximity to a tumor tissue and/or metastasis.

It has been found by the inventors that intratumoral application respectively administration of the RNA containing composition according to the invention is capable of effectively treating tumor and/or cancer diseases and related disorders. It has been shown that intratumoral application is surprisingly effective in decreasing tumor size. Moreover the application of the RNA containing composition according to the invention was able to increase survival in animal models.

The at least one RNA of the RNA containing composition may be selected from the group consisting of chemically modified or unmodified RNA, single-stranded or double-stranded RNA, coding or non-coding RNA, mRNA, oligoribonucleotide, viral RNA, retroviral RNA, replicon RNA, tRNA, rRNA, immunostimulatory RNA, microRNA, siRNA, small nuclear RNA (snRNA), small-hairpin (sh) RNA riboswitch, RNA aptamer, RNA decoy, antisense RNA, a ribozyme, or any combination thereof. In specific embodiments the at least one RNA of the RNA containing composition is selected from a coding RNA or a non-coding RNA.

Coding RNA:

According to a preferred embodiment of the invention the at least one RNA of the RNA containing composition comprises at least one coding region encoding at least one peptide or protein. Preferably, the coding RNA is selected from the group consisting of mRNA, viral RNA, retroviral RNA, and replicon RNA.

In preferred embodiments of the invention the at least one RNA of the RNA containing composition codes for at least one cytokine and/or for at least one chemokine and/or for at least one suicide gene product, and/or at least one immunogenic protein or peptide and/or for at least one cell death/apoptosis inducer and/or for at least one angiogenesis inhibitor and/or for at least one heat shock protein and/or for at least one tumor antigen and/or for at least one β-catenin inhibitor and/or for at least one activator of the STING (stimulator of interferon genes) pathway and/or at least one checkpoint modulator and/or at least one antibody, and/or at least one dominant negative receptor, and/or at least one decoy receptor, and/or at least one inhibitor of myeloid derived suppressor cells (MDSCs), and/or at least one IDO pathway inhibitor, and/or at least one protein or peptide that bind inhibitors of apoptosis, or fragments or variants thereof as will be outlined in more detail below.

1. Cytokines

In a preferred embodiment of the inventive RNA containing composition the RNA comprises at least one coding region that codes for at least one cytokine, or a fragment or variant thereof.

Preferably the cytokine is an interleukin (IL). One or more interleukins may be chosen e.g. from the following list: IL-1α, IL-1β, IL-1ra (antagonist), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10; IL-11, IL-12, IL-13, IL14, IL-15, IL-16, IL-17A, IL-17B, EL-17C, IL-17D, IL-17E, IL-17F, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28A/B, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35. Moreover the cytokine may be one or more cytokines chosen from the TNF family, e.g. chosen from the following list: TNF, especially TNFα, LTα, LTβ, LIGHT, TWEAK, APRIL, BAFF, TL1A, GITRL, OX40L, CD40L (CD154), FASL, CD27L, CD30L, 4-1BBL, TRAIL, RANK ligand. Further examples of preferred cytokines may be chosen from the following list: FLT3 ligand, G-CSF, GM-CSF, IFNα/β/ω, IFNγ, LIF, M-CSF, MIF, OSM, Stem Cell Factor, TGFβ1, TGFβ2, TGFβ3, TSLP ligand.

Particularly preferred are cytokines chosen from the following list: IL-12, IL-15, IL-2, IFNγ, TNFα, IL-18, IFNα, IL-1β, IL-32, IL-7, IL-21, IL-8, GM-CSF.

In an especially preferred embodiment of the invention the RNA of the inventive composition codes for Interleukin-12 or CD40L. It has been shown by the inventors, that mRNA coding for this cytokines is especially effective when applied in the inventive approach. Particularly preferred are RNA sequences according to SEQ ID Nos. 1, 3, 4194, 4195, 4196, 4197, 4198, 4199, 4200 encoding IL-12. Furthermore RNA sequences according to SEQ ID Nos. 3898, 3899, 3900, 3901, 3902, 3903, 3904, 10073, encoding CD40L are particularly preferred.

According to preferred embodiments in the context of the present invention cytokines may be selected from any cytokine selected from the group consisting of 4-1BBL; Apo2L/TRAIL; APRIL; BAFF; CD27L; CD30L; CD40L_(CD154); CXCL8; EL-17C; FasL; FLT3_ligand; G-CSF; GITRL; GM-CSF; IFNalpha; IFNB; IFNG; IFNomega; IL-1_alpha; IL-1_beta; IL-10; IL-11; IL-12; IL-12A; IL-13; IL-14; IL-15; IL-16; IL-17A; IL-17B; IL-17D; IL-17F; IL-18; IL-19; IL-1ra_(antagonist); IL-2; IL-20; IL-21; IL-22; IL-23; IL-24; IL-25; IL-26; IL-27A; IL-27B; IL-28A; IL-28B; IL-29; IL-3; IL-31; IL-32; IL-33; IL-37; IL-4; IL-5; IL-6; IL-7; IL-9; LIF; LIGHT; LTalpha; LTbeta; M-CSF; MIF; OSM; OX40L; RANK_ligand; Stem_Cell_Factor; TGFbeta1; TGFbeta2; TGFbeta3; TL1A; TNF; TWEAK, preferably as disclosed in Table 1. Particularly preferred in this context are the RNA sequences encoding a cytokine according to Table 1.

TABLE 1 Cytokines: RNA Protein Sequence Sequence wild type Optimized SEQ ID SEQ ID RNA Sequence Gene Name Protein Accession No. NO: NO: SEQ ID NO: 4-1BBL UniProtKB: P41273 3849 3850 3851, 3852, 3853, 3854, 3855, 3856 APRIL UniProtKB: O75888 3857 3858 3859, 3860, 3861, 3862, 3863, 3864 BAFF UniProtKB: Q5H8V1 3865 3866 3867, 3868, 3869, 3870, 3871, 3872 BAFF UniProtKB: Q9Y275 3873 3874 3875, 3876, 3877, 3878, 3879, 3880 CD27L UniProtKB: P32970 3881 3882 3883, 3884, 3885, 3886, 3887, 3888 CD30L UniProtKB: P32971 3889 3890 3891, 3892, 3893, 3894, 3895, 3896 CD40L_(CD154) UniProtKB: P29965 3897 3898 3899, 3900, 3901, 3902, 3903, 3904 EL-17C UniProtKB: Q9P0M4 3905 3906 3907, 3908, 3909, 3910, 3911, 3912 FLT3_ligand Genbank: AAA90950.1 3913 3914 3915, 3916, 3917, 3918, 3919, 3920 FLT3_ligand UniProtKB: P49771 3921 3922 3923, 3924, 3925, 3926, 3927, 3928 G-CSF UniProtKB: P09919 3929 3930 3931, 3932, 3933, 3934, 3935, 3936 GITRL UniProtKB: Q9UNG2 3937 3938 3939, 3940, 3941, 3942, 3943, 3944 GM-CSF UniProtKB: P04141 3945 3946 3947, 3948, 3949, 3950, 3951, 3952 IFNalpha UniProtKB: G9JKF1 3953 3954 3955, 3956, 3957, 3958, 3959, 3960 IFNalpha UniProtKB: P01562 3961 3962 3963, 3964, 3965, 3966, 3967, 3968 IFNalpha UniProtKB: P01563 3969 3970 3971, 3972, 3973, 3974, 3975, 3976 IFNalpha UniProtKB: P01566 3977 3978 3979, 3980, 3981, 3982, 3983, 3984 IFNalpha UniProtKB: P01567 3985 3986 3987, 3988, 3989, 3990, 3991, 3992 IFNalpha UniProtKB: P01568 3993 3994 3995, 3996, 3997, 3998, 3999, 4000 IFNalpha UniProtKB: P01569 4001 4002 4003, 4004, 4005, 4006, 4007, 4008 IFNalpha UniProtKB: P01570 4009 4010 4011, 4012, 4013, 4014, 4015, 4016 IFNalpha UniProtKB: P01571 4017 4018 4019, 4020, 4021, 4022, 4023, 4024 IFNalpha UniProtKB: P05013 4025 4026 4027, 4028, 4029, 4030, 4031, 4032 IFNalpha UniProtKB: P05014 4033 4034 4035, 4036, 4037, 4038, 4039, 4040 IFNalpha UniProtKB: P05015 4041 4042 4043, 4044, 4045, 4046, 4047, 4048 IFNalpha UniProtKB: P32881 4049 4050 4051, 4052, 4053, 4054, 4055, 4056 IFNalpha UniProtKB: Q14618 4057 4058 4059, 4060, 4061, 4062, 4063, 4064 IFNalpha UniProtKB: Q86UP4 4065 4066 4067, 4068, 4069, 4070, 4071, 4072 IFNB UniProtKB: P01574 4073 4074 4075, 4076, 4077, 4078, 4079, 4080 IFNB UniProtKB: Q15943 4081 4082 4083, 4084, 4085, 4086, 4087, 4088 IFNG UniProtKB: P01579 4089 4090 4091, 4092, 4093, 4094, 4095, 4096 IFNG UniProtKB: Q14609 4097 4098 4099, 4100, 4101, 4102, 4103, 4104 IFNG UniProtKB: Q14610 4105 4106 4107, 4108, 4109, 4110, 4111, 4112 IFNG UniProtKB: Q14611 4113 4114 4115, 4116, 4117, 4118, 4119, 4120 IFNG UniProtKB: Q14612 4121 4122 4123, 4124, 4125, 4126, 4127, 4128 IFNG UniProtKB: Q14613 4129 4130 4131, 4132, 4133, 4134, 4135, 4136 IFNG UniProtKB: Q14614 4137 4138 4139, 4140, 4141, 4142, 4143, 4144 IFNG UniProtKB: Q14615 4145 4146 4147, 4148, 4149, 4150, 4151, 4152 IFNG UniProtKB: Q8NHY9 4153 4154 4155, 4156, 4157, 4158, 4159, 4160 IFNomega UniProtKB: P05000 4161 4162 4163, 4164, 4165, 4166, 4167, 4168 IL-10 UniProtKB: P22301 4169 4170 4171, 4172, 4173, 4174, 4175, 4176 IL-11 UniProtKB: P20809 4177 4178 4179, 4180, 4181, 4182, 4183, 4184 IL-12A UniProtKB: P29459 4185 4186 4187, 4188, 4189, 4190, 4191, 4192 IL-12 UniProtKB: P29460 4193 4194 4195, 4196, 4197, 4198, 4199, 4200 IL-13 UniProtKB: P35225 4201 4202 4203, 4204, 4205, 4206, 4207, 4208 IL-14 UniProtKB: P40222 4209 4210 4211, 4212, 4213, 4214, 4215, 4216 IL-15 UniProtKB: P40933 4217 4218 4219, 4220, 4221, 4222, 4223, 4224 IL-16 UniProtKB: Q14005 4225 4226 4227, 4228, 4229, 4230, 4231, 4232 IL-17A UniProtKB: Q16552 4233 4234 4235, 4236, 4237, 4238, 4239, 4240 IL-17B UniProtKB: Q9NRM6 4241 4242 4243, 4244, 4245, 4246, 4247, 4248 IL-17B UniProtKB: Q9UHF5 4249 4250 4251, 4252, 4253, 4254, 4255, 4256 IL-17D UniProtKB: Q8TAD2 4257 4258 4259, 4260, 4261, 4262, 4263, 4264 IL-17F UniProtKB: F1JZ09 4265 4266 4267, 4268, 4269, 4270, 4271, 4272 IL-17F UniProtKB: Q96PD4 4273 4274 4275, 4276, 4277, 4278, 4279, 4280 IL-18 UniProtKB: A0A024R3E0 4281 4282 4283, 4284, 4285, 4286, 4287, 4288 IL-18 UniProtKB: B0YJ28 4289 4290 4291, 4292, 4293, 4294, 4295, 4296 IL-18 UniProtKB: Q14116 4297 4298 4299, 4300, 4301, 4302, 4303, 4304 IL-19 UniProtKB: Q9UHD0 4305 4306 4307, 4308, 4309, 4310, 4311, 4312 IL-1_alpha UniProtKB: P01583 4313 4314 4315, 4316, 4317, 4318, 4319, 4320 IL-1_beta UniProtKB: P01584 4321 4322 4323, 4324, 4325, 4326, 4327, 4328 IL-1ra_(antagonist) UniProtKB: P18510-2 4329 4330 4331, 4332, 4333, 4334, 4335, 4336 IL-1ra_(antagonist) UniProtKB: P18510-3 4337 4338 4339, 4340, 4341, 4342, 4343, 4344 IL-1ra_(antagonist) UniProtKB: P18510 4345 4346 4347, 4348, 4349, 4350, 4351, 4352 IL-20 UniProtKB: Q9NYY1 4353 4354 4355, 4356, 4357, 4358, 4359, 4360 IL-21 RefSeq: NP_001193935.1 4361 4362 4363, 4364, 4365, 4366, 4367, 4368 IL-21 RefSeq: NP_068575.1 4369 4370 4371, 4372, 4373, 4374, 4375, 4376 IL-22 UniProtKB: Q9GZX6 4377 4378 4379, 4380, 4381, 4382, 4383, 4384 IL-23 UniProtKB: Q9NPF7 4385 4386 4387, 4388, 4389, 4390, 4391, 4392 IL-24 UniProtKB: Q13007 4393 4394 4395, 4396, 4397, 4398, 4399, 4400 IL-24 UniProtKB: Q2YHE6 4401 4402 4403, 4404, 4405, 4406, 4407, 4408 IL-25 UniProtKB: Q969H8 4409 4410 4411, 4412, 4413, 4414, 4415, 4416 IL-25 UniProtKB: Q9H293 4417 4418 4419, 4420, 4421, 4422, 4423, 4424 IL-26 UniProtKB: Q9NPH9 4425 4426 4427, 4428, 4429, 4430, 4431, 4432 IL-27A UniProtKB: Q8NEV9 4433 4434 4435, 4436, 4437, 4438, 4439, 4440 IL-27B UniProtKB: Q14213 4441 4442 4443, 4444, 4445, 4446, 4447, 4448 IL-28A UniProtKB: Q8IZJ0 4449 4450 4451, 4452, 4453, 4454, 4455, 4456 IL-28B UniProtKB: Q8IZI9 4457 4458 4459, 4460, 4461, 4462, 4463, 4464 IL-29 UniProtKB: Q8IU54 4465 4466 4467, 4468, 4469, 4470, 4471, 4472 IL-2 UniProtKB: P60568 4473 4474 4475, 4476, 4477, 4478, 4479, 4480 IL-2 UniProtKB: Q0GK43 4481 4482 4483, 4484, 4485, 4486, 4487, 4488 IL-2 UniProtKB: Q13169 4489 4490 4491, 4492, 4493, 4494, 4495, 4496 IL-2 UniProtKB: Q6NZ91 4497 4498 4499, 4500, 4501, 4502, 4503, 4504 IL-2 UniProtKB: Q6NZ93 4505 4506 4507, 4508, 4509, 4510, 4511, 4512 IL-31 UniProtKB: Q6EBC2 4513 4514 4515, 4516, 4517, 4518, 4519, 4520 IL-32 UniProtKB: P24001 4521 4522 4523, 4524, 4525, 4526, 4527, 4528 IL-33 UniProtKB: O95760 4529 4530 4531, 4532, 4533, 4534, 4535, 4536 IL-37 UniProtKB: Q9NZH6 4537 4538 4539, 4540, 4541, 4542, 4543, 4544 IL-3 UniProtKB: P08700 4545 4546 4547, 4548, 4549, 4550, 4551, 4552 IL-3 UniProtKB: Q6NZ78 4553 4554 4555, 4556, 4557, 4558, 4559, 4560 IL-3 UniProtKB: Q6NZ79 4561 4562 4563, 4564, 4565, 4566, 4567, 4568 IL-4 UniProtKB: P05112-2 4569 4570 4571, 4572, 4573, 4574, 4575, 4576 IL-4 UniProtKB: P05112 4577 4578 4579, 4580, 4581, 4582, 4583, 4584 IL-5 UniProtKB: P05113 4585 4586 4587, 4588, 4589, 4590, 4591, 4592 IL-6 UniProtKB: P05231 4593 4594 4595, 4596, 4597, 4598, 4599, 4600 IL-7 UniProtKB: A8K673 4601 4602 4603, 4604, 4605, 4606, 4607, 4608 IL-7 UniProtKB: P13232 4609 4610 4611, 4612, 4613, 4614, 4615, 4616 IL-9 UniProtKB: P15248 4617 4618 4619, 4620, 4621, 4622, 4623, 4624 LIF UniProtKB: P15018 4625 4626 4627, 4628, 4629, 4630, 4631, 4632 LIGHT UniProtKB: O43557 4633 4634 4635, 4636, 4637, 4638, 4639, 4640 LTalpha UniProtKB: B4DVZ8 4641 4642 4643, 4644, 4645, 4646, 4647, 4648 LTalpha UniProtKB: P01374 4649 4650 4651, 4652, 4653, 4654, 4655, 4656 LTalpha UniProtKB: P09960 4657 4658 4659, 4660, 4661, 4662, 4663, 4664 LTalpha UniProtKB: Q5ST95 4665 4666 4667, 4668, 4669, 4670, 4671, 4672 LTalpha UniProtKB: Q5STV3 4673 4674 4675, 4676, 4677, 4678, 4679, 4680 LTalpha UniProtKB: Q6FG55 4681 4682 4683, 4684, 4685, 4686, 4687, 4688 LTbeta UniProtKB: Q06643 4689 4690 4691, 4692, 4693, 4694, 4695, 4696 LTbeta UniProtKB: Q5STB2 4697 4698 4699, 4700, 4701, 4702, 4703, 4704 M-CSF UniProtKB: P09603 4705 4706 4707, 4708, 4709, 4710, 4711, 4712 MIF UniProtKB: A6MUU8 4713 4714 4715, 4716, 4717, 4718, 4719, 4720 MIF UniProtKB: P14174 4721 4722 4723, 4724, 4725, 4726, 4727, 4728 OSM UniProtKB: P13725 4729 4730 4731, 4732, 4733, 4734, 4735, 4736 OX40L UniProtKB: P23510 4737 4738 4739, 4740, 4741, 4742, 4743, 4744 OX40L UniProtKB: P43489 4745 4746 4747, 4748, 4749, 4750, 4751, 4752 RANK_ligand UniProtKB: O14788 4753 4754 4755, 4756, 4757, 4758, 4759, 4760 Stem_Cell_Factor UniProtKB: P21583-2 4761 4762 4763, 4764, 4765, 4766, 4767, 4768 Stem_Cell_Factor UniProtKB: P21583 4769 4770 4771, 4772, 4773, 4774, 4775, 4776 TGFbeta1 UniProtKB: A0A024R0P8 4777 4778 4779, 4780, 4781, 4782, 4783, 4784 TGFbeta1 UniProtKB: P01137 4785 4786 4787, 4788, 4789, 4790, 4791, 4792 TGFbeta2 UniProtKB: P61812 4793 4794 4795, 4796, 4797, 4798, 4799, 4800 TGFbeta3 UniProtKB: A5YM40 4801 4802 4803, 4804, 4805, 4806, 4807, 4808 TGFbeta3 UniProtKB: P10600 4809 4810 4811, 4812, 4813, 4814, 4815, 4816 TL1A UniProtKB: O95150 4817 4818 4819, 4820, 4821, 4822, 4823, 4824 TWEAK UniProtKB: Q4ACW9 4825 4826 4827, 4828, 4829, 4830, 4831, 4832 CXCL8 UniProtKB: P10145 5265 5266 5267, 5268, 5269, 5270, 5271, 5272 Apo2L/TRAIL UniProtKB: P50591 6897 6898 6899, 6900, 6901, 6902, 6903, 6904 FasL UniProtKB: P48023 7321 7322 7323, 7324, 7325, 7326, 7327, 7328 TNF UniProtKB: P01375 7369 7370 7371, 7372, 7373, 7374, 7375, 7376 TNF UniProtKB: Q5STB3 7377 7378 7379, 7380, 7381, 7382, 7383, 7384

According to the present invention, in a more preferred embodiment, the inventive composition comprises at least one RNA, preferably an mRNA comprising at least one coding region encoding at least one cytokine or a fragment or variant thereof, wherein the at least one coding region comprises an RNA sequence being identical or at least 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the RNA sequences according to the SEQ ID Nos as disclosed in Table 1.

2. Chemokines:

In a further preferred embodiment of the inventive RNA containing composition the RNA comprises at least one coding region that codes for at least one chemokine, or a fragment or variant thereof.

Chemokines are chemotactic cytokines that control the migratory patterns and positioning of immune cells, as reviewed by Griffith et al., 2014. Annu. Rev. Immunol. 32:659-702 (PMID 24655300). Chemokine function is critical for all immune cell movement ranging from the migration required for immune cell development and homeostasis, to that required for the generation of primary and amnestic cellular and humoral immune responses, to the pathologic recruitment of immune cells in disease. Chemokines constitute the largest family of cytokines, consisting of approximately 50 endogenous chemokine ligands in humans and mice.

According to preferred embodiments in the context of the present invention chemokines may be selected from any chemokine selected from the group consisting of CCL1; CCL11; CCL12; CCL13; CCL14; CCL15; CCL16; CCL17; CCL18; CCL19; CCL2; CCL20; CCL21; CCL22; CCL24; CCL25; CCL26; CCL27; CCL28; CCL3; CCL4; CCL5; CCL6; CCL7; CCL8; CCL9; CX3CL1; CXCL1; CXCL10; CXCL11; CXCL12; CXCL13; CXCL14; CXCL15; CXCL2; CXCL3; CXCL4; CXCL5; CXCL6; CXCL7; CXCL8; CXCL9; XCL1; XCL2, preferably as disclosed in Table 2. Particularly preferred in this context are the RNA sequences encoding a chemokine according to Table 2.

TABLE 2 Chemokines RNA Protein Sequence Sequence wild type SEQ ID SEQ ID RNA Sequence Gene Name Protein Accession No. NO: NO: SEQ ID NO: CCL11 UniProtKB: P51671 4833 4834 4835, 4836, 4837, 4838, 4839, 4840 CCL11 UniProtKB: Q6I9T4 4841 4842 4843, 4844, 4845, 4846, 4847, 4848 CCL12 UniProtKB: Q62401 4849 4850 4851, 4852, 4853, 4854, 4855, 4856 CCL13 UniProtKB: Q99616 4857 4858 4859, 4860, 4861, 4862, 4863, 4864 CCL14 UniProtKB: Q16627 4865 4866 4867, 4868, 4869, 4870, 4871, 4872 CCL15 UniProtKB: Q16663 4873 4874 4875, 4876, 4877, 4878, 4879, 4880 CCL16 UniProtKB: O15467 4881 4882 4883, 4884, 4885, 4886, 4887, 4888 CCL17 UniProtKB: Q92583 4889 4890 4891, 4892, 4893, 4894, 4895, 4896 CCL18 UniProtKB: P55774 4897 4898 4899, 4900, 4901, 4902, 4903, 4904 CCL19 UniProtKB: Q6IBD6 4905 4906 4907, 4908, 4909, 4910, 4911, 4912 CCL19 UniProtKB: Q99731 4913 4914 4915, 4916, 4917, 4918, 4919, 4920 CCL1 UniProtKB: P22362 4921 4922 4923, 4924, 4925, 4926, 4927, 4928 CCL20 UniProtKB: P78556 4929 4930 4931, 4932, 4933, 4934, 4935, 4936 CCL21 UniProtKB: O00585 4937 4938 4939, 4940, 4941, 4942, 4943, 4944 CCL22 UniProtKB: O00626 4945 4946 4947, 4948, 4949, 4950, 4951, 4952 CCL24 UniProtKB: O00175 4953 4954 4955, 4956, 4957, 4958, 4959, 4960 CCL25 UniProtKB: O15444 4961 4962 4963, 4964, 4965, 4966, 4967, 4968 CCL26 UniProtKB: Q9Y258 4969 4970 4971, 4972, 4973, 4974, 4975, 4976 CCL27 UniProtKB: Q5VZ77 4977 4978 4979, 4980, 4981, 4982, 4983, 4984 CCL28 UniProtKB: A0N0Q3 4985 4986 4987, 4988, 4989, 4990, 4991, 4992 CCL28 UniProtKB: Q9NRJ3 4993 4994 4995, 4996, 4997, 4998, 4999, 5000 CCL2 UniProtKB: P13500 5001 5002 5003, 5004, 5005, 5006, 5007, 5008 CCL3 UniProtKB: A0N0R1 5009 5010 5011, 5012, 5013, 5014, 5015, 5016 CCL3 UniProtKB: P10147 5017 5018 5019, 5020, 5021, 5022, 5023, 5024 CCL4 UniProtKB: P13236 5025 5026 5027, 5028, 5029, 5030, 5031, 5032 CCL4 UniProtKB: Q7M4M2 5033 5034 5035, 5036, 5037, 5038, 5039, 5040 CCL5 UniProtKB: D0EI67 5041 5042 5043, 5044, 5045, 5046, 5047, 5048 CCL5 UniProtKB: P13501 5049 5050 5051, 5052, 5053, 5054, 5055, 5056 CCL6 UniProtKB: P27784 5057 5058 5059, 5060, 5061, 5062, 5063, 5064 CCL7 UniProtKB: P80098 5065 5066 5067, 5068, 5069, 5070, 5071, 5072 CCL7 UniProtKB: Q7Z7Q8 5073 5074 5075, 5076, 5077, 5078, 5079, 5080 CCL8 UniProtKB: H0UIC7 5081 5082 5083, 5084, 5085, 5086, 5087, 5088 CCL8 UniProtKB: P80075 5089 5090 5091, 5092, 5093, 5094, 5095, 5096 CCL9 UniProtKB: P51670 5097 5098 5099, 5100, 5101, 5102, 5103, 5104 CX3CL1 UniProtKB: A0N0N7 5105 5106 5107, 5108, 5109, 5110, 5111, 5112 CX3CL1 UniProtKB: P78423 5113 5114 5115, 5116, 5117, 5118, 5119, 5120 CX3CL1 UniProtKB: Q6I9S9 5121 5122 5123, 5124, 5125, 5126, 5127, 5128 CXCL10 UniProtKB: A0A024RDA4 5129 5130 5131, 5132, 5133, 5134, 5135, 5136 CXCL10 UniProtKB: P02778 5137 5138 5139, 5140, 5141, 5142, 5143, 5144 CXCL11 UniProtKB: O14625 5145 5146 5147, 5148, 5149, 5150, 5151, 5152 CXCL12 UniProtKB: P48061 5153 5154 5155, 5156, 5157, 5158, 5159, 5160 CXCL13 UniProtKB: L8E878 5161 5162 5163, 5164, 5165, 5166, 5167, 5168 CXCL13 UniProtKB: O43927 5169 5170 5171, 5172, 5173, 5174, 5175, 5176 CXCL14 UniProtKB: O95715 5177 5178 5179, 5180, 5181, 5182, 5183, 5184 CXCL15 UniProtKB: Q9WVL7 5185 5186 5187, 5188, 5189, 5190, 5191, 5192 CXCL1 UniProtKB: P09341 5193 5194 5195, 5196, 5197, 5198, 5199, 5200 CXCL2 UniProtKB: A0A024RDD9 5201 5202 5203, 5204, 5205, 5206, 5207, 5208 CXCL2 UniProtKB: P19875 5209 5210 5211, 5212, 5213, 5214, 5215, 5216 CXCL3 UniProtKB: P19876 5217 5218 5219, 5220, 5221, 5222, 5223, 5224 CXCL4 UniProtKB: P02776 5225 5226 5227, 5228, 5229, 5230, 5231, 5232 CXCL5 UniProtKB: P42830 5233 5234 5235, 5236, 5237, 5238, 5239, 5240 CXCL5 UniProtKB: Q6I9S7 5241 5242 5243, 5244, 5245, 5246, 5247, 5248 CXCL6 UniProtKB: P80162 5249 5250 5251, 5252, 5253, 5254, 5255, 5256 CXCL7 UniProtKB: P02775 5257 5258 5259, 5260, 5261, 5262, 5263, 5264 CXCL8 UniProtKB: P10145 5265 5266 5267, 5268, 5269, 5270, 5271, 5272 CXCL9 UniProtKB: L8E8X0 5273 5274 5275, 5276, 5277, 5278, 5279, 5280 CXCL9 UniProtKB: Q07325 5281 5282 5283, 5284, 5285, 5286, 5287, 5288 XCL1 UniProtKB: P47992 5289 5290 5291, 5292, 5293, 5294, 5295, 5296 XCL2 UniProtKB: Q9UBD3 5297 5298 5299, 5300, 5301, 5302, 5303, 5304

In this context particularly preferred are chemokines chosen from the following list: CXCL9, CXCL10, CCL5, XCL1, CCL20, CCL19, CCL21, CCL2, CCL3, CCL16, and CXCL12.

According to the present invention, in a more preferred embodiment, the inventive composition comprises at least one RNA, preferably an mRNA comprising at least one coding region encoding at least one chemokine or a fragment or variant thereof, wherein the at least one coding region comprises an RNA sequence being identical or at least 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the RNA sequences according to the SEQ ID Nos as disclosed in Table 2.

3. Suicide Gene Products

In a further preferred embodiment of the inventive RNA containing composition the RNA codes for at least one so-called suicide gene product, especially for a suicide enzyme, preferably for a nucleotide metabolizing enzyme. Preferably the RNA is used in combination with a prodrug which is a substrate of the suicide gene product, especially the suicide enzyme, and which is converted to a cytotoxic compound by the suicide gene product. The appropriate prodrug may be added to the inventive RNA composition or it may be administered separately to the patient.

One or more preferred suicide enzymes may be chosen from the following list: thymidine kinase, preferably a viral thymidine kinase, more preferrably Herpes simplex virus thymidine kinase, Varicella zoster thymidine kinase; a plant thymidine kinase, preferably a tomato thymidine kinase; cytosine deaminase, preferably bacterial cytosine deaminase or Yeast cytosine deaminase; deoxynucleoside kinase, preferably Drosophila melanogaster deoxynucleoside kinase; deoxycytidine kinase, preferably a mammalian deoxycytidine kinase, purine nucleoside phosphorylase, preferably a bacterial purine nucleoside phosphorylase.

It is already known that suicide gene therapy is a promising treatment for cancer (Ardiani et al., 2012. Curr. Gene Ther. 12(2):77-91. PMID: 22384805). This approach is based on the successful delivery and expression of the suicide gene in tumor cells. The suicide gene encodes an enzyme with the unique ability to activate an otherwise ineffective prodrug. Following suicide gene expression in transfected cells, an appropriate prodrug is administered and is converted to a cytotoxic compound by the actions of the suicide gene product. As most suicide genes encode enzymes belonging to the class of nucleotide metabolizing enzymes, the general mode of action of activated prodrugs is interference with DNA synthesis that consequently results in induction of apoptosis. The potency of these drugs is maximized in cancer cells due to their greater proliferative rate as compared to normal cells. Because of the prospect to preferentially deliver genes to tumor cells, this strategy has the potential to offer selective tumor killing while sparing normal cells, a feature that standard chemotherapeutic and radiotherapy approaches do not generally afford.

The following table 3 (Ardiani et al., 2012. Curr. Gene Ther. 12(2):77-91. PMID: 22384805) summarizes preferred nucleotide metabolizing enzymes usable in the inventive approach. The table includes variants and mutants of such enzymes which were generated by protein engineering strategies.

TABLE 3 Suicide enzymes Drug Natural Variants/ inhibitors Enzyme Source gene substrate Prodrug Mutants action* Herpes Herpes Thymidine Ganciclovir (GCV), Mutant 30 1 Simplex Simplex Virus 1 acyclovir (ACV) Mutant 75 1 Virus (HSV-1) SR39 1 Thymidine Thymidine A168H 1 Kinase Kinase (TK) A167Y 1 (HSVTK) Q125N 1, 2 Bacterial Escherichia coli - Cytosine 5-Fluorocytosine D314 1, 2, 4 Cytosine codA (5-FC) mutants Deaminase bCD1525 1, 2, 4 (bCD) Yeast Saccharomyces Cytosine 5-FC yCD triple 1, 2, 4 Cytosine cerevisiae - D92E 1, 2, 4 Deaminase fcy1 (yCD) Drosophila Drosophila melanogaster - All four azidothymidine MuD 1, 5 melanogaster dNK deoxy-ribo- (AZT), B5 1 Deoxynucleoside nucleo- dideoxycytoinse B10 1, 3 Kinase sides (ddC); M88R 1 (Dm-dNK) CdA; HDHD-12, 1, 5 9-beta-D- HD-16 arabinofuranosyl- R4.V3 1 2-fluoroadenine (F-AraA); GCV, 9-beta-D- arabinosylguanine (AraG); 2′,3′- didehydro-3′- deoxythymidine (D4T); 2′,3′- Dideoxythymidine (ddT) Deoxy- Homo sapiens - Deoxy- 2′,2′-difluoro- DMMA, 1, 3 cytidine dCK cytidine deoxycytidine DMLA Kinase (dCK) (dFdC), AraA, β-L- EpTK6 1, 3, 5 thymidine (L-dT) Ser-74 1, 3 AZT cytarabine 5′- monophosphate (AraC) Purine Escherichia coli - Purine ribo- 9-(6-deoxy-α-L- M64V 1, 4 Nucleoside deoD nucleo- talofuranosyl)-6- Phosphor- sides methylpurine ylase (PNP) (Me(talo)-MeP-R) *Drug inhibitory action. 1: DNA synthesis; 2: Thymidylate synthetase; 3: Ribonucleotide reductase; 4: RNA/protein synthesis; 5: Reverse transcriptase.

Herpes simplex virus type 1 thymidine kinase (HSVTK, EC 2.7.1.21), a homodimer with a subunit molecular mass of 45 kDa, is responsible for the phosphorylation of thymidine, deoxycytidine, deoxythymidylate (dTMP) as well as various pharmaceutically important pyrimidine and guanosine analogs. Of particular note, HSVTK is responsible for the initial and rate limiting phosphorylation of the antiviral guanosine analogs acyclovir (ACV) and ganciclovir (GCV). Once monophosphorylated these analogs can be further phosphorylated by endogenous enzymes (guanylate kinase and nucleoside diphosphokinase) before being incorporated into nascent DNA to cause double strand destabilization and, subsequently, cell death.

Moreover, the Varicella zoster virus thymidine kinase (VZV-tk) may be used e.g. in conjunction with the prodrug 6-methoxypurine arabinoside (ara-M) or 1-(2′-deoxy-2-flioro-b-D-arabinofuranosyl)-5-iodouracil (FIAU). Other examples are thymidine kinases of Aleutian disease virus (ADV), respiratory syncytial virus (RSV) and cytomegalovirus (CMV).

Cytosine deaminase (CD; EC 3.5.4.1) is an enzyme in the pyrimidine salvage pathway that catalyzes the deamination of cytosine to form uracil and ammonia. CD from E. coli (bCD) is a hexamer of 48 kDA subunits with a catalytic metal iron. This enzyme is absent in mammals and uniquely present in fungi and bacteria. It is used in suicide gene therapy because of its ability to deaminate the anti-fungal drug, 5-fluorocytosine (5FC), to 5-fluorouracil (5FU), a potent anti-neoplastic drug. UPRT (Uracil phosphoribosyltransferase) may be used as potential enhancer.

Saccharomyces cerevisiae or Yeast cytosine deaminase (yCD, EC 3.5.4.1) is a homodimer of 17.5 kDa subunits and has been shown to be more active towards 5FC than bCD (22-fold lower K_(m)) with a slightly better catalytic efficiency (k_(cat)/K_(m)) toward 5FC relative to its natural substrate cytosine.

Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK; EC 2.7.1.145) is a 29 kDa homodimeric, multisubstrate kinase able to phosphorylate all four natural deoxyribonucleosides required for DNA synthesis. In addition to its broad substrate specificity, Dm-dNK exhibits higher catalytic rates toward these natural deoxynucleosides and several nucleoside analogs as compared to mammalian deoxynucleoside kinases. Due to these distinctive characteristics Dm-dNK is a especially preferred enzyme for the inventive suicide gene therapy application.

Human deoxycytidine kinase (dCK; EC 2.7.1.74) is a 30.5 kDa homodimeric enzyme in the salvage pathway of deoxyribonucleosides and is responsible for activating all natural deoxyribonucleosides, excluding thymidine, as precursors for DNA synthesis. Due to its broad substrate specificity, dCK is able to activate multiple nucleoside analogs effective against different types of cancer. However, wild type dCK is intrinsically a relatively poor catalyst with low turnover rates and prodrug activation is dependent on its expression levels. Indeed, nucleoside analogs that are efficient substrates of dCK, such as cytarabine (AraC), fludarabine (F-AraA), cladribine (CdA), and gemcitabine (dFdC), are effective anti-leukemic agents as lymphoblasts have been shown to have high dCK expression levels whereas cancer cells lacking dCK activity are resistant to these same analogs. Therefore dCK is an especially preferred enzyme for the inventive approach.

Preferably the RNA of the inventive RNA containing composition is used in combination with further components which enhance the cytotoxic effect of the treatment. It is especially preferred to use the RNA in combination with RNA coding for connexins and/or with a protein of the connexin family or parts or fragments thereof. Connexins are transmembrane proteins which form gap junctions between cells. They allow transfer of e.g. molecules between neighboring cells thereby enabling the transfer of cytoxic substances.

Although suicide gene therapy is a fairly new anti-cancer approach, the concept was originally described more than two decades ago in 1986 by Moolten (Moolten, 1986. Cancer Res. 46(10):5276-81). He also proposed the existence of what is currently known as the bystander effect, now widely recognized as a fundamental feature of suicide gene therapy success. By definition the bystander effect is the extension of cytotoxic effects from transfected cells to non-transfected neighboring cells such that complete tumor regression is observed when only a small subpopulation of tumor cells is successfully transfected. This phenomenon is crucial to the overall effectiveness of suicide gene therapy today due to low transfection efficiencies achievable by available delivery systems.

The bystander effect is thought to occur via two major mechanisms: local and immune-mediated. The local mechanism involves the killing of untransfected nearby cells due to the transfer of toxic materials or suicide enzymes through gap junctions, apoptotic vesicles or through diffusion of soluble toxic metabolites. Gap junctions are important in cell-cell interactions and are responsible for the transfer of ions, nucleotides and small molecules to adjacent cells. The transfer of toxic drugs through gap junctions, however, may not be available in certain types of tumors that down regulate intracellular gap junction communication and display disorganized and non-functional gap junctions. To address this problem, several studies have increased the expression of connexins, the building blocks of gap junctions, and demonstrated that enhanced bystander and cell killing effects were achieved.

Accumulating evidence from in vivo experiments suggests that the immune-mediated bystander effect plays an important role in tumor regression as well. The presence of inflammatory infiltrates, chemokines, and cytokines have been found elevated in regressing tumors of immune competent animals receiving suicide gene therapy treatment. These cytokines and chemokines further induce the production of immune-regulatory molecules able to stimulate a more robust anti-cancer effect and additionally, because death of transfected cells is through apoptosis, numerous inflammatory signals may be released to evoke a potent immune response. Therefore the combination of the inventive composition with connexins or with RNA coding for connexins is especially preferred, because it strengthens the bystander effect thereby increasing the efficiency of the inventive RNA containing composition.

According to preferred embodiments in the context of the present invention suicide gene products may be selected from any suicide gene product selected from the group consisting of Cytosine_Deaminase_codA; Cytosine_Deaminase_fcy1; Deoxy-cytidine_Kinase_dCK; Deoxynucleoside_Kinase_dNK; Purine_Nucleoside_Phosphorylase_deoD; Thymidine_Kinase_TK, preferably as disclosed in Table 4. Particularly preferred in this context are the RNA sequences encoding a suicide gene product according to Table 4.

TABLE 4 Suicide Gene Products RNA Protein Sequence RNA Protein Sequence wild type Sequence Gene Name Accession No. SEQ ID NO: SEQ ID NO: SEQ ID NO: Cytosine_Deaminase_codA UniProtKB: 5305 5306 5307, 5308, 5309, A0A024KS17 5310, 5311, 5312 Cytosine_Deaminase_codA UniProtKB: 5313 5314 5315, 5316, 5317, A0A0H2V4N7 5318, 5319, 5320 Cytosine_Deaminase_codA UniProtKB: 5321 5322 5323, 5324, 5325, A0A0H2YX33 5326, 5327, 5328 Cytosine_Deaminase_codA UniProtKB: 5329 5330 5331, 5332, 5333, F4NM90 5334, 5335, 5336 Cytosine_Deaminase_codA UniProtKB: 5337 5338 5339, 5340, 5341, H9UNZ4 5342, 5343, 5344 Cytosine_Deaminase_codA UniProtKB: 5345 5346 5347, 5348, 5349, Q1RFJ5 5350, 5351, 5352 Cytosine_Deaminase_codA UniProtKB: 5353 5354 5355, 5356, 5357, Q2VP09 5358, 5359, 5360 Cytosine_Deaminase_codA UniProtKB: 5361 5362 5363, 5364, 5365, Q53ZC8 5366, 5367, 5368 Cytosine_Deaminase_codA UniProtKB: 5369 5370 5371, 5372, 5373, Q6Q8Q1 5374, 5375, 5376 Cytosine_Deaminase_codA UniProtKB: 5377 5378 5379, 5380, 5381, W8ZNH5 5382, 5383, 5384 Cytosine_Deaminase_fcy1 UniProtKB: 5385 5386 5387, 5388, 5389, A0A023ZJG6 5390, 5391, 5392 Cytosine_Deaminase_fcy1 UniProtKB: 5393 5394 5395, 5396, 5397, A0A024XGF7 5398, 5399, 5400 Cytosine_Deaminase_fcy1 UniProtKB: 5401 5402 5403, 5404, 5405, A0A024XUW9 5406, 5407, 5408 Cytosine_Deaminase_fcy1 UniProtKB: 5409 5410 5411, 5412, 5413, A0A0C5ITD0 5414, 5415, 5416 Cytosine_Deaminase_fcy1 UniProtKB: 5417 5418 5419, 5420, 5421, A0A0D4WVI5 5422, 5423, 5424 Cytosine_Deaminase_fcy1 UniProtKB: 5425 5426 5427, 5428, 5429, A0A0D4WY08 5430, 5431, 5432 Cytosine_Deaminase_fcy1 UniProtKB: 5433 5434 5435, 5436, 5437, A0A0D4WZA2 5438, 5439, 5440 Cytosine_Deaminase_fcy1 UniProtKB: 5441 5442 5443, 5444, 5445, A0A0D4WZQ5 5446, 5447, 5448 Cytosine_Deaminase_fcy1 UniProtKB: 5449 5450 5451, 5452, 5453, A0A0D4X0R8 5454, 5455, 5456 Cytosine_Deaminase_fcy1 UniProtKB: 5457 5458 5459, 5460, 5461, A0A0D4X195 5462, 5463, 5464 Cytosine_Deaminase_fcy1 UniProtKB: 5465 5466 5467, 5468, 5469, A0A0D4X2R9 5470, 5471, 5472 Cytosine_Deaminase_fcy1 UniProtKB: 5473 5474 5475, 5476, 5477, A0A0D4X3Q1 5478, 5479, 5480 Cytosine_Deaminase_fcy1 UniProtKB: 5481 5482 5483, 5484, 5485, A0A0D4X4K1 5486, 5487, 5488 Cytosine_Deaminase_fcy1 UniProtKB: 5489 5490 5491, 5492, 5493, A0A0D4X5B7 5494, 5495, 5496 Cytosine_Deaminase_fcy1 UniProtKB: 5497 5498 5499, 5500, 5501, A0A0D4X7R4 5502, 5503, 5504 Cytosine_Deaminase_fcy1 UniProtKB: 5505 5506 5507, 5508, 5509, A0A0D4X7X4 5510, 5511, 5512 Cytosine_Deaminase_fcy1 UniProtKB: 5513 5514 5515, 5516, 5517, A0A0D4XA07 5518, 5519, 5520 Cytosine_Deaminase_fcy1 UniProtKB: 5521 5522 5523, 5524, 5525, A0A0D4XA25 5526, 5527, 5528 Cytosine_Deaminase_fcy1 UniProtKB: 5529 5530 5531, 5532, 5533, A0A0D4XAV6 5534, 5535, 5536 Cytosine_Deaminase_fcy1 UniProtKB: 5537 5538 5539, 5540, 5541, A0A0D4XCJ5 5542, 5543, 5544 Cytosine_Deaminase_fcy1 UniProtKB: 5545 5546 5547, 5548, 5549, A0A0D4XDL4 5550, 5551, 5552 Cytosine_Deaminase_fcy1 UniProtKB: 5553 5554 5555, 5556, 5557, A0A0D4XG53 5558, 5559, 5560 Cytosine_Deaminase_fcy1 UniProtKB: 5561 5562 5563, 5564, 5565, A0A0D4XGH3 5566, 5567, 5568 Cytosine_Deaminase_fcy1 UniProtKB: 5569 5570 5571, 5572, 5573, A0A0D4XHD4 5574, 5575, 5576 Cytosine_Deaminase_fcy1 UniProtKB: 5577 5578 5579, 5580, 5581, A0A0D4XIK5 5582, 5583, 5584 Cytosine_Deaminase_fcy1 UniProtKB: 5585 5586 5587, 5588, 5589, A0A0D4XJR4 5590, 5591, 5592 Cytosine_Deaminase_fcy1 UniProtKB: 5593 5594 5595, 5596, 5597, A0A0D4XL36 5598, 5599, 5600 Cytosine_Deaminase_fcy1 UniProtKB: 5601 5602 5603, 5604, 5605, A0A0D4XNH2 5606, 5607, 5608 Cytosine_Deaminase_fcy1 UniProtKB: 5609 5610 5611, 5612, 5613, A0A0D4XNS1 5614, 5615, 5616 Cytosine_Deaminase_fcy1 UniProtKB: 5617 5618 5619, 5620, 5621, A0A0D4XQY5 5622, 5623, 5624 Cytosine_Deaminase_fcy1 UniProtKB: 5625 5626 5627, 5628, 5629, A0A0D4XS80 5630, 5631, 5632 Cytosine_Deaminase_fcy1 UniProtKB: 5633 5634 5635, 5636, 5637, A0A0D4XS82 5638, 5639, 5640 Cytosine_Deaminase_fcy1 UniProtKB: 5641 5642 5643, 5644, 5645, A0A0D4XTC2 5646, 5647, 5648 Cytosine_Deaminase_fcy1 UniProtKB: 5649 5650 5651, 5652, 5653, A0A0D4XUZ4 5654, 5655, 5656 Cytosine_Deaminase_fcy1 UniProtKB: 5657 5658 5659, 5660, 5661, A0A0D4XW26 5662, 5663, 5664 Cytosine_Deaminase_fcy1 UniProtKB: 5665 5666 5667, 5668, 5669, A0A0D4XXD1 5670, 5671, 5672 Cytosine_Deaminase_fcy1 UniProtKB: 5673 5674 5675, 5676, 5677, A0A0D4XYH3 5678, 5679, 5680 Cytosine_Deaminase_fcy1 UniProtKB: 5681 5682 5683, 5684, 5685, A0A0D4XZT0 5686, 5687, 5688 Cytosine_Deaminase_fcy1 UniProtKB: 5689 5690 5691, 5692, 5693, A0A0D4Y164 5694, 5695, 5696 Cytosine_Deaminase_fcy1 UniProtKB: 5697 5698 5699, 5700, 5701, A0A0D4Y2A8 5702, 5703, 5704 Cytosine_Deaminase_fcy1 UniProtKB: 5705 5706 5707, 5708, 5709, A0A0D4Y3N1 5710, 5711, 5712 Cytosine_Deaminase_fcy1 UniProtKB: 5713 5714 5715, 5716, 5717, A0A0D4Y5S3 5718, 5719, 5720 Cytosine_Deaminase_fcy1 UniProtKB: 5721 5722 5723, 5724, 5725, A0A0D4Y5Y1 5726, 5727, 5728 Cytosine_Deaminase_fcy1 UniProtKB: 5729 5730 5731, 5732, 5733, A0A0D4Y7I2 5734, 5735, 5736 Cytosine_Deaminase_fcy1 UniProtKB: 5737 5738 5739, 5740, 5741, A0A0D4Y8S5 5742, 5743, 5744 Cytosine_Deaminase_fcy1 UniProtKB: 5745 5746 5747, 5748, 5749, A0A0D4YAR2 5750, 5751, 5752 Cytosine_Deaminase_fcy1 UniProtKB: 5753 5754 5755, 5756, 5757, A0A0D4YBY2 5758, 5759, 5760 Cytosine_Deaminase_fcy1 UniProtKB: 5761 5762 5763, 5764, 5765, A0A0D4YCB3 5766, 5767, 5768 Cytosine_Deaminase_fcy1 UniProtKB: 5769 5770 5771, 5772, 5773, A0A0D4YEC2 5774, 5775, 5776 Cytosine_Deaminase_fcy1 UniProtKB: 5777 5778 5779, 5780, 5781, A0A0D4YF30 5782, 5783, 5784 Cytosine_Deaminase_fcy1 UniProtKB: 5785 5786 5787, 5788, 5789, A0A0D4YGU2 5790, 5791, 5792 Cytosine_Deaminase_fcy1 UniProtKB: 5793 5794 5795, 5796, 5797, A0A0D4YHN3 5798, 5799, 5800 Cytosine_Deaminase_fcy1 UniProtKB: 5801 5802 5803, 5804, 5805, A0A0D4YIU4 5806, 5807, 5808 Cytosine_Deaminase_fcy1 UniProtKB: 5809 5810 5811, 5812, 5813, A0A0D4YJ74 5814, 5815, 5816 Cytosine_Deaminase_fcy1 UniProtKB: 5817 5818 5819, 5820, 5821, A0A0D4YKC5 5822, 5823, 5824 Cytosine_Deaminase_fcy1 UniProtKB: 5825 5826 5827, 5828, 5829, A0A0D4YMN8 5830, 5831, 5832 Cytosine_Deaminase_fcy1 UniProtKB: 5833 5834 5835, 5836, 5837, A0A0D4YMV6 5838, 5839, 5840 Cytosine_Deaminase_fcy1 UniProtKB: 5841 5842 5843, 5844, 5845, A0A0D4YPP6 5846, 5847, 5848 Cytosine_Deaminase_fcy1 UniProtKB: 5849 5850 5851, 5852, 5853, A0A0D4YRD4 5854, 5855, 5856 Cytosine_Deaminase_fcy1 UniProtKB: 5857 5858 5859, 5860, 5861, A0A0D4YS13 5862, 5863, 5864 Cytosine_Deaminase_fcy1 UniProtKB: 5865 5866 5867, 5868, 5869, A0A0D4YTJ7 5870, 5871, 5872 Cytosine_Deaminase_fcy1 UniProtKB: 5873 5874 5875, 5876, 5877, A0A0D4YUX9 5878, 5879, 5880 Cytosine_Deaminase_fcy1 UniProtKB: 5881 5882 5883, 5884, 5885, A0A0D4YV34 5886, 5887, 5888 Cytosine_Deaminase_fcy1 UniProtKB: 5889 5890 5891, 5892, 5893, A0A0D4YXE1 5894, 5895, 5896 Cytosine_Deaminase_fcy1 UniProtKB: 5897 5898 5899, 5900, 5901, A0A0D4YYM6 5902, 5903, 5904 Cytosine_Deaminase_fcy1 UniProtKB: 5905 5906 5907, 5908, 5909, A0A0D4YZB7 5910, 5911, 5912 Cytosine_Deaminase_fcy1 UniProtKB: 5913 5914 5915, 5916, 5917, A0A0D4Z060 5918, 5919, 5920 Cytosine_Deaminase_fcy1 UniProtKB: 5921 5922 5923, 5924, 5925, A0A0D4Z1S2 5926, 5927, 5928 Cytosine_Deaminase_fcy1 UniProtKB: 5929 5930 5931, 5932, 5933, A0A0D4Z2L6 5934, 5935, 5936 Cytosine_Deaminase_fcy1 UniProtKB: 5937 5938 5939, 5940, 5941, A0A0D4Z4A1 5942, 5943, 5944 Cytosine_Deaminase_fcy1 UniProtKB: 5945 5946 5947, 5948, 5949, A0A0D4Z552 5950, 5951, 5952 Cytosine_Deaminase_fcy1 UniProtKB: 5953 5954 5955, 5956, 5957, A0A0D4Z6N6 5958, 5959, 5960 Cytosine_Deaminase_fcy1 UniProtKB: 5961 5962 5963, 5964, 5965, A0A0D4Z800 5966, 5967, 5968 Cytosine_Deaminase_fcy1 UniProtKB: 5969 5970 5971, 5972, 5973, A0A0D4Z9V2 5974, 5975, 5976 Cytosine_Deaminase_fcy1 UniProtKB: 5977 5978 5979, 5980, 5981, A0A0D4ZB52 5982, 5983, 5984 Cytosine_Deaminase_fcy1 UniProtKB: 5985 5986 5987, 5988, 5989, A0A0D4ZCA2 5990, 5991, 5992 Cytosine_Deaminase_fcy1 UniProtKB: 5993 5994 5995, 5996, 5997, A0A0D4ZCG3 5998, 5999, 6000 Cytosine_Deaminase_fcy1 UniProtKB: 6001 6002 6003, 6004, 6005, A0A0D4ZEM2 6006, 6007, 6008 Cytosine_Deaminase_fcy1 UniProtKB: 6009 6010 6011, 6012, 6013, A0A0D4ZFD0 6014, 6015, 6016 Cytosine_Deaminase_fcy1 UniProtKB: 6017 6018 6019, 6020, 6021, A0A0D4ZGR1 6022, 6023, 6024 Cytosine_Deaminase_fcy1 UniProtKB: 6025 6026 6027, 6028, 6029, A0A0D4ZIM2 6030, 6031, 6032 Cytosine_Deaminase_fcy1 UniProtKB: 6033 6034 6035, 6036, 6037, A0A0D4ZJC0 6038, 6039, 6040 Cytosine_Deaminase_fcy1 UniProtKB: 6041 6042 6043, 6044, 6045, A0A0D4ZK17 6046, 6047, 6048 Cytosine_Deaminase_fcy1 UniProtKB: 6049 6050 6051, 6052, 6053, A0A0D4ZMC8 6054, 6055, 6056 Cytosine_Deaminase_fcy1 UniProtKB: 6057 6058 6059, 6060, 6061, A0A0D4ZMX9 6062, 6063, 6064 Cytosine_Deaminase_fcy1 UniProtKB: 6065 6066 6067, 6068, 6069, A0A0D4ZP21 6070, 6071, 6072 Cytosine_Deaminase_fcy1 UniProtKB: 6073 6074 6075, 6076, 6077, A0A0D4ZQ62 6078, 6079, 6080 Cytosine_Deaminase_fcy1 UniProtKB: 6081 6082 6083, 6084, 6085, A0A0D4ZQ92 6086, 6087, 6088 Cytosine_Deaminase_fcy1 UniProtKB: 6089 6090 6091, 6092, 6093, A0A0D4ZS31 6094, 6095, 6096 Cytosine_Deaminase_fcy1 UniProtKB: 6097 6098 6099, 6100, 6101, A0A0D4ZS87 6102, 6103, 6104 Cytosine_Deaminase_fcy1 UniProtKB: 6105 6106 6107, 6108, 6109, A0A0D4ZTS6 6110, 6111, 6112 Cytosine_Deaminase_fcy1 UniProtKB: 6113 6114 6115, 6116, 6117, A0A0D4ZUK0 6118, 6119, 6120 Cytosine_Deaminase_fcy1 UniProtKB: 6121 6122 6123, 6124, 6125, A0A0D4ZVN6 6126, 6127, 6128 Cytosine_Deaminase_fcy1 UniProtKB: 6129 6130 6131, 6132, 6133, A0A0D4ZWP2 6134, 6135, 6136 Cytosine_Deaminase_fcy1 UniProtKB: 6137 6138 6139, 6140, 6141, A0A0D4ZX07 6142, 6143, 6144 Cytosine_Deaminase_fcy1 UniProtKB: 6145 6146 6147, 6148, 6149, Q12178 6150, 6151, 6152 Cytosine_Deaminase_fcy1 UniProtKB: 6153 6154 6155, 6156, 6157, W7PK48 6158, 6159, 6160 Cytosine_Deaminase_fcy1 UniProtKB: 6161 6162 6163, 6164, 6165, W7R647 6166, 6167, 6168 Deoxy-cytidine_Kinase_dCK UniProtKB: 6169 6170 6171, 6172, 6173, P27707 6174, 6175, 6176 Deoxynucleoside_Kinase_dNK UniProtKB: 6177 6178 6179, 6180, 6181, Q540Z9 6182, 6183, 6184 Deoxynucleoside_Kinase_dNK UniProtKB: 6185 6186 6187, 6188, 6189, Q9XZT6 6190, 6191, 6192 Purine_Nucleoside_(—) UniProtKB: 6193 6194 6195, 6196, 6197, Phosphorylase_deoD A0A023Z7B9 6198, 6199, 6200 Purine_Nucleoside_(—) UniProtKB: 6201 6202 6203, 6204, 6205, Phosphorylase_deoD A0A024KMI2 6206, 6207, 6208 Purine_Nucleoside_(—) UniProtKB: 6209 6210 6211, 6212, 6213, Phosphorylase_deoD A0A0E0SRY5 6214, 6215, 6216 Purine_Nucleoside_(—) UniProtKB: 6217 6218 6219, 6220, 6221, Phosphorylase_deoD A0A0E0U7I4 6222, 6223, 6224 Purine_Nucleoside_(—) UniProtKB: 6225 6226 6227, 6228, 6229, Phosphorylase_deoD A0A0E0VFI3 6230, 6231, 6232 Purine_Nucleoside_(—) UniProtKB: 6233 6234 6235, 6236, 6237, Phosphorylase_deoD A0A0E0Y455 6238, 6239, 6240 Purine_Nucleoside_(—) UniProtKB: 6241 6242 6243, 6244, 6245, Phosphorylase_deoD A0A0E1M7E2 6246, 6247, 6248 Purine_Nucleoside_(—) UniProtKB: 6249 6250 6251, 6252, 6253, Phosphorylase_deoD A0A0E3KJD7 6254, 6255, 6256 Purine_Nucleoside_(—) UniProtKB: 6257 6258 6259, 6260, 6261, Phosphorylase_deoD A0A0F6CCW6 6262, 6263, 6264 Purine_Nucleoside_(—) UniProtKB: 6265 6266 6267, 6268, 6269, Phosphorylase_deoD A0A0F6FGI8 6270, 6271, 6272 Purine_Nucleoside_(—) UniProtKB: 6273 6274 6275, 6276, 6277, Phosphorylase_deoD A0A0F6GWR2 6278, 6279, 6280 Purine_Nucleoside_(—) UniProtKB: 6281 6282 6283, 6284, 6285, Phosphorylase_deoD A0A0G2SIK5 6286, 6287, 6288 Purine_Nucleoside_(—) UniProtKB: 6289 6290 6291, 6292, 6293, Phosphorylase_deoD A0A0G3J9R6 6294, 6295, 6296 Purine_Nucleoside_(—) UniProtKB: 6297 6298 6299, 6300, 6301, Phosphorylase_deoD A0A0G3J9Y2 6302, 6303, 6304 Purine_Nucleoside_(—) UniProtKB: 6305 6306 6307, 6308, 6309, Phosphorylase_deoD A0A0G3KD68 6310, 6311, 6312 Purine_Nucleoside_(—) UniProtKB: 6313 6314 6315, 6316, 6317, Phosphorylase_deoD A0A0H2Z6H1 6318, 6319, 6320 Purine_Nucleoside_(—) UniProtKB: 6321 6322 6323, 6324, 6325, Phosphorylase_deoD A0A0H3EQW1 6326, 6327, 6328 Purine_Nucleoside_(—) UniProtKB: 6329 6330 6331, 6332, 6333, Phosphorylase_deoD A0A0H3XF09 6334, 6335, 6336 Purine_Nucleoside_(—) UniProtKB: 6337 6338 6339, 6340, 6341, Phosphorylase_deoD A0A0J9WZC9 6342, 6343, 6344 Purine_Nucleoside_(—) UniProtKB: 6345 6346 6347, 6348, 6349, Phosphorylase_deoD A7ZVS7 6350, 6351, 6352 Purine_Nucleoside_(—) UniProtKB: 6353 6354 6355, 6356, 6357, Phosphorylase_deoD A8A8B3 6358, 6359, 6360 Purine_Nucleoside_(—) UniProtKB: 6361 6362 6363, 6364, 6365, Phosphorylase_deoD B1IS35 6366, 6367, 6368 Purine_Nucleoside_(—) UniProtKB: 6369 6370 6371, 6372, 6373, Phosphorylase_deoD B1LEI9 6374, 6375, 6376 Purine_Nucleoside_(—) UniProtKB: 6377 6378 6379, 6380, 6381, Phosphorylase_deoD B1XFJ4 6382, 6383, 6384 Purine_Nucleoside_(—) UniProtKB: 6385 6386 6387, 6388, 6389, Phosphorylase_deoD B3HEI4 6390, 6391, 6392 Purine_Nucleoside_(—) UniProtKB: 6393 6394 6395, 6396, 6397, Phosphorylase_deoD B5Z4R6 6398, 6399, 6400 Purine_Nucleoside_(—) UniProtKB: 6401 6402 6403, 6404, 6405, Phosphorylase_deoD B6I6N1 6406, 6407, 6408 Purine_Nucleoside_(—) UniProtKB: 6409 6410 6411, 6412, 6413, Phosphorylase_deoD B7LEN0 6414, 6415, 6416 Purine_Nucleoside_(—) UniProtKB: 6417 6418 6419, 6420, 6421, Phosphorylase_deoD B7LXU6 6422, 6423, 6424 Purine_Nucleoside_(—) UniProtKB: 6425 6426 6427, 6428, 6429, Phosphorylase_deoD B7MNJ1 6430, 6431, 6432 Purine_Nucleoside_(—) UniProtKB: 6433 6434 6435, 6436, 6437, Phosphorylase_deoD B7N2V8 6438, 6439, 6440 Purine_Nucleoside_(—) UniProtKB: 6441 6442 6443, 6444, 6445, Phosphorylase_deoD B7NH52 6446, 6447, 6448 Purine_Nucleoside_(—) UniProtKB: 6449 6450 6451, 6452, 6453, Phosphorylase_deoD B7NW64 6454, 6455, 6456 Purine_Nucleoside_(—) UniProtKB: 6457 6458 6459, 6460, 6461, Phosphorylase_deoD B7UR12 6462, 6463, 6464 Purine_Nucleoside_(—) UniProtKB: 6465 6466 6467, 6468, 6469, Phosphorylase_deoD C3SE47 6470, 6471, 6472 Purine_Nucleoside_(—) UniProtKB: 6473 6474 6475, 6476, 6477, Phosphorylase_deoD C4ZT66 6478, 6479, 6480 Purine_Nucleoside_(—) UniProtKB: 6481 6482 6483, 6484, 6485, Phosphorylase_deoD C8TQD7 6486, 6487, 6488 Purine_Nucleoside_(—) UniProtKB: 6489 6490 6491, 6492, 6493, Phosphorylase_deoD C8U157 6494, 6495, 6496 Purine_Nucleoside_(—) UniProtKB: 6497 6498 6499, 6500, 6501, Phosphorylase_deoD C8UN92 6502, 6503, 6504 Purine_Nucleoside_(—) UniProtKB: 6505 6506 6507, 6508, 6509, Phosphorylase_deoD D3GY24 6510, 6511, 6512 Purine_Nucleoside_(—) UniProtKB: 6513 6514 6515, 6516, 6517, Phosphorylase_deoD D3QNE6 6518, 6519, 6520 Purine_Nucleoside_(—) UniProtKB: 6521 6522 6523, 6524, 6525, Phosphorylase_deoD D6I4N2 6526, 6527, 6528 Purine_Nucleoside_(—) UniProtKB: 6529 6530 6531, 6532, 6533, Phosphorylase_deoD D6IHU2 6534, 6535, 6536 Purine_Nucleoside_(—) UniProtKB: 6537 6538 6539, 6540, 6541, Phosphorylase_deoD D6J6A4 6542, 6543, 6544 Purine_Nucleoside_(—) UniProtKB: 6545 6546 6547, 6548, 6549, Phosphorylase_deoD E0J437 6550, 6551, 6552 Purine_Nucleoside_(—) UniProtKB: 6553 6554 6555, 6556, 6557, Phosphorylase_deoD E2QLE4 6558, 6559, 6560 Purine_Nucleoside_(—) UniProtKB: 6561 6562 6563, 6564, 6565, Phosphorylase_deoD E3PFG7 6566, 6567, 6568 Purine_Nucleoside_(—) UniProtKB: 6569 6570 6571, 6572, 6573, Phosphorylase_deoD E8YEH0 6574, 6575, 6576 Purine_Nucleoside_(—) UniProtKB: 6577 6578 6579, 6580, 6581, Phosphorylase_deoD F4NLK2 6582, 6583, 6584 Purine_Nucleoside_(—) UniProtKB: 6585 6586 6587, 6588, 6589, Phosphorylase_deoD F4SEX7 6590, 6591, 6592 Purine_Nucleoside_(—) UniProtKB: 6593 6594 6595, 6596, 6597, Phosphorylase_deoD F4STB8 6598, 6599, 6600 Purine_Nucleoside_(—) UniProtKB: 6601 6602 6603, 6604, 6605, Phosphorylase_deoD F4T9F1 6606, 6607, 6608 Purine_Nucleoside_(—) UniProtKB: 6609 6610 6611, 6612, 6613, Phosphorylase_deoD F4UXB7 6614, 6615, 6616 Purine_Nucleoside_(—) UniProtKB: 6617 6618 6619, 6620, 6621, Phosphorylase_deoD F4VN60 6622, 6623, 6624 Purine_Nucleoside_(—) UniProtKB: 6625 6626 6627, 6628, 6629, Phosphorylase_deoD F4VQF8 6630, 6631, 6632 Purine_Nucleoside_(—) UniProtKB: 6633 6634 6635, 6636, 6637, Phosphorylase_deoD H9V0H4 6638, 6639, 6640 Purine_Nucleoside_(—) UniProtKB: 6641 6642 6643, 6644, 6645, Phosphorylase_deoD J7QV83 6646, 6647, 6648 Purine_Nucleoside_(—) UniProtKB: 6649 6650 6651, 6652, 6653, Phosphorylase_deoD P0ABP8 6654, 6655, 6656 Purine_Nucleoside_(—) UniProtKB: 6657 6658 6659, 6660, 6661, Phosphorylase_deoD Q0T8S9 6662, 6663, 6664 Purine_Nucleoside_(—) UniProtKB: 6665 6666 6667, 6668, 6669, Phosphorylase_deoD Q1R259 6670, 6671, 6672 Purine_Nucleoside_(—) UniProtKB: 6673 6674 6675, 6676, 6677, Phosphorylase_deoD W8ZSE4 6678, 6679, 6680 Purine_Nucleoside_(—) UniProtKB: 6681 6682 6683, 6684, 6685, Phosphorylase_deoD X5FDR9 6686, 6687, 6688 Thymidine_Kinase_TK UniProtKB: 6689 6690 6691, 6692, 6693, B2CPP5 6694, 6695, 6696 Thymidine_Kinase_TK UniProtKB: 6697 6698 6699, 6700, 6701, B2CPP6 6702, 6703, 6704 Thymidine_Kinase_TK UniProtKB: 6705 6706 6707, 6708, 6709, B2CPP7 6710, 6711, 6712 Thymidine_Kinase_TK UniProtKB: 6713 6714 6715, 6716, 6717, B2CPP8 6718, 6719, 6720 Thymidine_Kinase_TK UniProtKB: 6721 6722 6723, 6724, 6725, B2CPP9 6726, 6727, 6728 Thymidine_Kinase_TK UniProtKB: 6729 6730 6731, 6732, 6733, B2CPQ0 6734, 6735, 6736 Thymidine_Kinase_TK UniProtKB: 6737 6738 6739, 6740, 6741, B2CPQ2 6742, 6743, 6744 Thymidine_Kinase_TK UniProtKB: 6745 6746 6747, 6748, 6749, B2CPQ3 6750, 6751, 6752 Thymidine_Kinase_TK UniProtKB: 6753 6754 6755, 6756, 6757, B2CPQ4 6758, 6759, 6760 Thymidine_Kinase_TK UniProtKB: 6761 6762 6763, 6764, 6765, B2CPQ5 6766, 6767, 6768 Thymidine_Kinase_TK UniProtKB: 6769 6770 6771, 6772, 6773, O72346 6774, 6775, 6776 Thymidine_Kinase_TK UniProtKB: 6777 6778 6779, 6780, 6781, P06478 6782, 6783, 6784 Thymidine_Kinase_TK UniProtKB: 6785 6786 6787, 6788, 6789, P08333 6790, 6791, 6792 Thymidine_Kinase_TK UniProtKB: 6793 6794 6795, 6796, 6797, Q9DLP2 6798, 6799, 6800 Thymidine_Kinase_TK UniProtKB: 6801 6802 6803, 6804, 6805, Q9ENS0 6806, 6807, 6808 Thymidine_Kinase_TK UniProtKB: 6809 6810 6811, 6812, 6813, Q9ENS1 6814, 6815, 6816 Thymidine_Kinase_TK UniProtKB: 6817 6818 6819, 6820, 6821, Q9ENS2 6822, 6823, 6824 Thymidine_Kinase_TK UniProtKB: 6825 6826 6827, 6828, 6829, Q9ENS3 6830, 6831, 6832 Thymidine_Kinase_TK UniProtKB: 6833 6834 6835, 6836, 6837, Q9ENS4 6838, 6839, 6840 Thymidine_Kinase_TK UniProtKB: 6841 6842 6843, 6844, 6845, Q9ENS5 6846, 6847, 6848 Thymidine_Kinase_TK UniProtKB: 6849 6850 6851, 6852, 6853, Q9IYZ7 6854, 6855, 6856 Thymidine_Kinase_TK UniProtKB: 6857 6858 6859, 6860, 6861, Q9IYZ9 6862, 6863, 6864 Thymidine_Kinase_TK UniProtKB: 6865 6866 6867, 6868, 6869, Q9IZ02 6870, 6871, 6872 Thymidine_Kinase_TK UniProtKB: 6873 6874 6875, 6876, 6877, Q9IZ03 6878, 6879, 6880 Thymidine_Kinase_TK UniProtKB: 6881 6882 6883, 6884, 6885, Q9IZ07 6886, 6887, 6888 Thymidine_Kinase_TK UniProtKB: 6889 6890 6891, 6892, 6893, Q9QNF7 6894, 6895, 6896

According to the present invention, in a more preferred embodiment, the inventive composition comprises at least one RNA, preferably an mRNA comprising at least one coding region encoding at least one suicide gene product or a fragment or variant thereof, wherein the at least one coding region comprises an RNA sequence being identical or at least 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the RNA sequences according to the SEQ ID Nos as disclosed in Table 4.

4. Immunogenic Proteins or Peptides

Preferably the RNA, preferably mRNA of the inventive RNA composition codes for at least one immunogenic protein or peptide, especially a protein or peptide of a pathogen, preferably a viral pathogen, or a fragment or variant thereof. By using RNA which codes for an immunogenic protein or peptide which is preferably a pathogenic antigen it is possible to utilize preexisting immunity against such antigens for treatment of tumor and/or cancer diseases. The memory immune response is triggered and the immune system is strengthened for attacking tumor cells.

This embodiment of the invention is based on the recognition that in principle every organism with an immune system exhibits “memory immune responses” against certain foreign molecules (antigens), for example proteins, in particular viral or bacterial proteins. If an organism has already been infected at an earlier point in time with the antigen an immune response against e.g. the viral protein has already been triggered by this infection. The immune system has a “memory” of this response and stores it. As consequence of a reinfection with the antigen the immune response is reactivated. Such reactivation may proceed by administration of an RNA, preferably mRNA coding for the antigen, wherein the preferred intratumoral administration according to the invention is especially effective. By reactivation of the memory immune response against e.g. viral pathogens it is possible to destroy tumor cells effectively.

Preferred examples of immunogenic proteins or peptides for this embodiment of the invention are proteins or peptides of widespread pathogens, i.e. pathogens with which every organism, in particular mammals, preferably humans, has a high probability of being infected at least once in his/her lifetime. These include, for example, any structural or non-structural protein or peptide of:

-   -   influenza virus type A or B or any other orthomyxovirus         (influenza type C),     -   picornaviruses, such as rhinovirus or hepatitis A virus,     -   togaviruses, such as alphavirus or rubivirus, e.g. Sindbis,         Semliki-Forest or rubeolavirus (measles virus),     -   rubella virus (German measles virus),     -   coronaviruses, in particular subtypes HCV-229E or HCV-OC43,     -   rhabdoviruses, such as rabies virus,     -   paramyxoviruses, such as mumps virus,     -   reoviruses, such as group A, B or C rotavirus,     -   hepadnaviruses, such as hepatitis B virus,     -   papoviruses, such as human papillomaviruses (HPV) of any         serotype, especially from 1 to 75,     -   adenoviruses, in particular type 1 to 47,     -   herpesviruses, such as Herpes simplex virus 1, 2 or 3,     -   cytomegalovirus (CMV), preferably CMVpp65,     -   Epstein Barr virus (EBV),     -   vaccinia viruses and     -   the bacterium Chiamydophila pneumoniae (Chlamydia pneumoniae).

Further examples of preferred immunogenic proteins or peptides are proteins or peptides of pathogens which only seldom infect an organism. Nevertheless RNA coding for one or more of these proteins or peptides may be effective in the inventive approach. These proteins or peptide include, for example, any structural or non-structural protein or peptide of:

-   -   Flaviviruses, such as dengue virus type 1 to 4, yellow fever         virus, West Nile virus, Japanese encephalitis virus     -   hepatitis C virus,     -   caliciviruses,     -   filoviruses, such as Ebola virus,     -   bornaviruses,     -   bunyaviruses, such as Rift Valley fever virus,     -   arenaviruses, such as LCMV (lymphocytic choriomeningitis virus)         or hemorrhagic fever viruses,     -   retroviruses, such as HIV and     -   parvoviruses.

Preferably the RNA of the inventive mRNA composition codes for influenza nucleoprotein (NP). It has been shown by the inventors that the use of a composition containing mRNA coding for influenza nucleoprotein is especially effective in reducing tumor size, when applied according to the inventive approach. In this context an mRNA encoding an Influenza nucleoprotein according to SEQ ID NO. 6 is particularly preferred.

5. Cell Death Inducers and Apoptosis Inducers:

In the broadest sense, an apoptosis inducer or cell death inducer has to be understood as a molecule inducing autophagy, cornification, excitotoxicity, necrosis, Wallerian degeneration, entosis, mitotic catastrophe, necroptosis and pyroptosis (reviewed in Kroemer, G., et al. “Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009.” Cell Death & Differentiation 16.1 (2009): 3-11.).

In a further preferred embodiment of the inventive RNA containing composition the RNA codes for at least one apoptosis inducer, preferably an apoptosis inducer chosen from the group consisting of the Bcl-2 family and tumor suppressor protein p53 and ligands of transmembrane death receptors, especially the TNF (tumor necrosis factor) receptor gene superfamily, pro-apoptic receptor agonists and Beclin-1.

A particularly preferred apoptosis inducer in the context of the present invention is Beclin-1 (derived from the BECN1 gene). It is known in the art that Beclin-1 interacts with Bcl-2, BCL2L2, GOPC and MAP1LC3A to regulate autophagy and cell death.

Apoptosis provides an important barrier against cancer. However, specific mutations (e.g. mutation of the tumor suppressor gene p53) enable some tumor cells to escape apoptotic death and become more malignant. By using an mRNA coding for at least one apoptosis inducer it is possible to reactivate apoptosis which is an important and effective system of the organism to eliminate cancer cells.

Preferred examples of apoptosis inducers may be chosen from the following list: Bcl-10, Bax, Bak, Bid, Bad, Bim, Bik, Blk, Cytochrome c, Caspases, especially Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Death domain, especially of Fas, preferably FasL, TNFα, Apo2L/TRAIL, agonist of DR4 and/or DR5, Apo3L, DR4 agonistic antibody, DR5 agonistic antibody, protein kinase R (PKR) (preferably constitutive active PKR), Granzyme B.

Two signalling pathways initiate apoptosis: the intrinsic pathway acts through intracellular Bcl-2 proteins, the extrinsic pathway through cell-surface pro-apoptotic receptors.

The intrinsic signaling pathway for programmed cell death involves non-receptor-mediated intracellular signals, inducing activities in the mitochondria that initiate apoptosis. Stimuli for the intrinsic pathway include viral infections or damage to the cell by toxins, free radicals, or radiation. Damage to the cellular DNA can also induce the activation of the intrinsic pathway for programmed cell death. These stimuli induce changes in the inner mitochondrial membrane that result in the loss of transmembrane potential, causing the release of pro-apoptotic proteins into the cytosol. Pro-apoptotic proteins activate caspases that mediate the destruction of the cell through many pathways. These proteins also translocate into the cellular nucleus, inducing DNA fragmentation, a hallmark of apoptosis. The regulation of pro-apoptotic events in the mitochondria occurs through activity of members of the Bcl-2 family of proteins and the tumor suppressor protein p53. Members of the Bcl-2 family of proteins may be pro-apoptotic or anti-apoptotic. The anti-apoptotic proteins are Bcl-2, Bcl-x, Bcl-xL, Bcl-XS, Bcl-w, and BAG. Pro-apoptotic proteins include Bcl-10, Bax, Bak, Bid, Bad, Bim, Bik, and Blk (Elmore, 2007. Toxicol Pathol. 35(4):495-516 (PMID: 17562483)), which are especially preferred for the inventive approach.

The extrinsic signaling pathway leading to apoptosis involves transmembrane death receptors that are members of the tumor necrosis factor (TNF) receptor gene superfamily. Members of this receptor family bind to extrinsic ligands and transduce intracellular signals that ultimately result in the destruction of the cell. The most well characterized ligands of these receptors to date are FasL, TNFα, Apo2L, and Apo3L. Corresponding receptors are FasR, TNFR1, DR3, and DR4/DR5. Molecules that stimulate the activity of these pro-apoptotic proteins or activate these receptors are currently under evaluation for their therapeutic potential in the treatment of cancer, including hematologic malignancies (Elmore, 2007. Toxicol Pathol. 35(4):495-516 (PMID: 17562483)). These extrinsic ligands are further especially preferred examples for use in the inventive approach.

New molecular insights have inspired the development of pro-apoptotic receptor agonists (PARAs), including the recombinant human protein apoptosis ligand 2/TNF-related apoptosis-inducing ligand (Apo2L/TRAIL). In addition, agonistic monoclonal antibodies to its signalling receptors DR4 (TRAILR1) and DR5 (TRAILR2) are under development. Mapatumumab is an example of a DR4 agonist antibody. Examples of DR5 agonistic antibodies include Lexatumumab, Apomab, AMG655, CS-1008 and LBY-135 (Ashkenazi, 2008. Nat. Rev. Drug Discov. 7(12):1001-12 (PMID: 18989337)).

The following table 5 summarizes some preferred apoptosis inducers.

TABLE 5 Apoptosis inducers Gene/Agent Example Intrinsic pathway Bcl-10 Bax Bak Bid Bad Bim Bik Blk Cytochrome c Caspase 3, 6, 7, 8, 9 Extrinsic pathway FasL TNFα Apo2L/TRAIL Apo3L DR4 agonist antibody Mapatumumab DR5 agonist antibody Lexatumumab, Apomab, AMG655, CS-1008, LBY-135 Other Granzyme B

According to preferred embodiments in the context of the present invention apoptosis inducers may be selected from any apoptosis inducer selected from the group consisting of Apo2L/TRAIL; Apo3L; Bad; Bak; Bax; Bcl-10; Bid; Bik; Bim; Blk; Caspase_3; Caspase_6; Caspase_7; Caspase_8; Caspase_9; Cytochrome_c; FasL; Granzyme_B; TNF, preferably as disclosed in Table 6. Particularly preferred in this context are the RNA sequences encoding an apoptosis inducer according to Table 6.

TABLE 6 Apoptosis inducers: RNA Optimized Protein Sequence RNA Sequence wild type Sequence Gene Name Protein Accession No. SEQ ID NO: SEQ ID NO: SEQ ID NO: Apo2L/TRAIL UniProtKB: P50591 6897 6898 6899, 6900, 6901, 6902, 6903, 6904 Apo3L UniProtKB: O43508 6905 6906 6907, 6908, 6909, 6910, 6911, 6912 Bad UniProtKB: A0A024R562 6913 6914 6915, 6916, 6917, 6918, 6919, 6920 Bad UniProtKB: Q92934 6921 6922 6923, 6924, 6925, 6926, 6927, 6928 Bak UniProtKB: Q16611 6929 6930 6931, 6932, 6933, 6934, 6935, 6936 Bak UniProtKB: Q8NFF3 6937 6938 6939, 6940, 6941, 6942, 6943, 6944 Bax UniProtKB: A0A0C4MVT1 6945 6946 6947, 6948, 6949, 6950, 6951, 6952 Bax UniProtKB: A0A0C4MW46 6953 6954 6955, 6956, 6957, 6958, 6959, 6960 Bax UniProtKB: A0A0C4MWS3 6961 6962 6963, 6964, 6965, 6966, 6967, 6968 Bax UniProtKB: I6LPK7 6969 6970 6971, 6972, 6973, 6974, 6975, 6976 Bax UniProtKB: K4JQN1 6977 6978 6979, 6980, 6981, 6982, 6983, 6984 Bax UniProtKB: Q07812 6985 6986 6987, 6988, 6989, 6990, 6991, 6992 Bcl-10 UniProtKB: O95999 6993 6994 6995, 6996, 6997, 6998, 6999, 7000 Bid UniProtKB: A8ASI8 7001 7002 7003, 7004, 7005, 7006, 7007, 7008 Bid UniProtKB: B2ZP78 7009 7010 7011, 7012, 7013, 7014, 7015, 7016 Bid UniProtKB: B2ZP79 7017 7018 7019, 7020, 7021, 7022, 7023, 7024 Bid UniProtKB: P55957 7025 7026 7027, 7028, 7029, 7030, 7031, 7032 Bik UniProtKB: A0A024R4X6 7033 7034 7035, 7036, 7037, 7038, 7039, 7040 Bik UniProtKB: Q13323 7041 7042 7043, 7044, 7045, 7046, 7047, 7048 Bim UniProtKB: O43521 7049 7050 7051, 7052, 7053, 7054, 7055, 7056 Blk UniProtKB: P51451 7057 7058 7059, 7060, 7061, 7062, 7063, 7064 Caspase_3 UniProtKB: P42574 7065 7066 7067, 7068, 7069, 7070, 7071, 7072 Caspase_6 UniProtKB: P55212 7073 7074 7075, 7076, 7077, 7078, 7079, 7080 Caspase_7 UniProtKB: P55210 7081 7082 7083, 7084, 7085, 7086, 7087, 7088 Caspase_8 UniProtKB: B5BU46 7089 7090 7091, 7092, 7093, 7094, 7095, 7096 Caspase_8 UniProtKB: B6CGU5 7097 7098 7099, 7100, 7101, 7102, 7103, 7104 Caspase_8 UniProtKB: C3S3G0 7105 7106 7107, 7108, 7109, 7110, 7111, 7112 Caspase_8 UniProtKB: Q14790 7113 7114 7115, 7116, 7117, 7118, 7119, 7120 Caspase_9 UniProtKB: A0A024R8F1 7121 7122 7123, 7124, 7125, 7126, 7127, 7128 Caspase_9 UniProtKB: A0A024R8I4 7129 7130 7131, 7132, 7133, 7134, 7135, 7136 Caspase_9 UniProtKB: P55211 7137 7138 7139, 7140, 7141, 7142, 7143, 7144 Caspase_9 UniProtKB: Q9H257 7145 7146 7147, 7148, 7149, 7150, 7151, 7152 Cytochrome_c UniProtKB: A0A024R9B7 7153 7154 7155, 7156, 7157, 7158, 7159, 7160 Cytochrome_c UniProtKB: A0A024RAP6 7161 7162 7163, 7164, 7165, 7166, 7167, 7168 Cytochrome_c UniProtKB: A0A024RBN6 7169 7170 7171, 7172, 7173, 7174, 7175, 7176 Cytochrome_c UniProtKB: A0A024RBY9 7177 7178 7179, 7180, 7181, 7182, 7183, 7184 Cytochrome_c UniProtKB: B8XYC5 7185 7186 7187, 7188, 7189, 7190, 7191, 7192 Cytochrome_c UniProtKB: G4XXL9 7193 7194 7195, 7196, 7197, 7198, 7199, 7200 Cytochrome_c UniProtKB: H0UI06 7201 7202 7203, 7204, 7205, 7206, 7207, 7208 Cytochrome_c UniProtKB: H6SG12 7209 7210 7211, 7212, 7213, 7214, 7215, 7216 Cytochrome_c UniProtKB: H6SG13 7217 7218 7219, 7220, 7221, 7222, 7223, 7224 Cytochrome_c UniProtKB: H6SG14 7225 7226 7227, 7228, 7229, 7230, 7231, 7232 Cytochrome_c UniProtKB: H6SG15 7233 7234 7235, 7236, 7237, 7238, 7239, 7240 Cytochrome_c UniProtKB: O95101 7241 7242 7243, 7244, 7245, 7246, 7247, 7248 Cytochrome_c UniProtKB: P08574 7249 7250 7251, 7252, 7253, 7254, 7255, 7256 Cytochrome_c UniProtKB: P99999 7257 7258 7259, 7260, 7261, 7262, 7263, 7264 Cytochrome_c UniProtKB: Q496I0 7265 7266 7267, 7268, 7269, 7270, 7271, 7272 Cytochrome_c UniProtKB: Q53XN1 7273 7274 7275, 7276, 7277, 7278, 7279, 7280 Cytochrome_c UniProtKB: Q6FGA0 7281 7282 7283, 7284, 7285, 7286, 7287, 7288 Cytochrome_c UniProtKB: Q6FGI7 7289 7290 7291, 7292, 7293, 7294, 7295, 7296 Cytochrome_c UniProtKB: Q71U45 7297 7298 7299, 7300, 7301, 7302, 7303, 7304 Cytochrome_c UniProtKB: Q86WV2 7305 7306 7307, 7308, 7309, 7310, 7311, 7312 Cytochrome_c UniProtKB: Q9UEG9 7313 7314 7315, 7316, 7317, 7318, 7319, 7320 FasL UniProtKB: P48023 7321 7322 7323, 7324, 7325, 7326, 7327, 7328 Granzyme_B UniProtKB: J3KQ52 7329 7330 7331, 7332, 7333, 7334, 7335, 7336 Granzyme_B UniProtKB: Q67BC3 7337 7338 7339, 7340, 7341, 7342, 7343, 7344 Granzyme_B UniProtKB: Q6XGZ2 7345 7346 7347, 7348, 7349, 7350, 7351, 7352 Granzyme_B UniProtKB: Q6XGZ3 7353 7354 7355, 7356, 7357, 7358, 7359, 7360 Granzyme_B UniProtKB: Q6XGZ4 7361 7362 7363, 7364, 7365, 7366, 7367, 7368 TNF UniProtKB: P01375 7369 7370 7371, 7372, 7373, 7374, 7375, 7376 TNF UniProtKB: Q5STB3 7377 7378 7379, 7380, 7381, 7382, 7383, 7384

According to the present invention, in a more preferred embodiment, the inventive composition comprises at least one RNA, preferably an mRNA comprising at least one coding region encoding at least one apoptosis inducer or cell death inducer or a fragment or variant thereof, wherein the at least one coding region comprises an RNA sequence being identical or at least 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the RNA sequences according to the SEQ ID Nos as disclosed in Table 6.

6. Angiogenesis Inhibitors

In a further preferred embodiment of the inventive RNA containing composition the at least one RNA, preferably mRNA codes for at least one angiogenesis modulator or inhibitor, preferably an endogenous angiogenesis inhibitor or a fragment or variant thereof. Tumor growth and survival depend on angiogenesis to provide a path for delivery of oxygen and nutrients to tumor cells. By using RNA coding for at least one angiogenesis inhibitor according to the inventive approach it is possible to block angiogenesis in a localized manner, namely within the tumor tissue, thereby providing an effective method for stopping tumor growth and decreasing tumor volume. Preferred examples of angiogenesis inhibitors according to the invention may be chosen from the following list: interferon alpha (IFN-α), (interferon beta) IFN-β, interferon gamma (IFN-γ), CXCL9, CXCL10, interleukin 12 (IL-12), platelet factor 4 (PF-4), tumor necrosis factor alpha (TNF-α), soluble fms-like tyrosine kinase 1 (sFLT-1), Fetal Liver Kinase 1 (FLK-1), Angiostatin, Endostatin, Vasostatin, Canstatin, Tumstatin, 16 kD prolacin fragment, tissue inhibitor of metalloproteinases 1 (TIMP-1), tissue inhibitor of metalloproteinases 2 (TIMP-2), tissue inhibitor of metalloproteinases 3 (TIMP-3), thrombospondin 1 (TSP-1), thrombospondin 2 (TSP-2), Maspin, PEX, soluble Tyrosine-protein kinase receptor 1 (sTie1), soluble Angiopoietin-1 receptor 2 (sTie2), Angiopoietin-1, Angiopoietin-2, Antivascular endothelial growth factor receptor 2 (VEGFR2) antibody (e.g. Alacizumab, Ramucirumab), Anti-vascular endothelial growth factor (VEGF) antibody (e.g. Brolucizumab, Ranibizumab, Bevacizumab), and Anti-vascular endothelial growth factor receptor 1 (VEGFR1) antibody (e.g. Icrucumab).

Without this process of blood vessel recruitment, tumor growth is limited to 1 to 2 mm², the diffusion limit of oxygen. Already in 1971, Folkman proposed that tumor growth could be arrested by blocking angiogenesis (Folkman, 1972. N. Engl. J. Med. 285(21):1182-6).

Angiogenesis is a multistep process of new blood vessel formation from preexisting vasculature that includes the activation, proliferation and migration of endothelial cells (ECs), disruption of vascular basement membranes, remodeling of the extracellular matrix of tissues, formation of vascular tubes and networks, recruitment of supporting cells, including smooth muscle cells and pericytes, and connection to the pre-existing vascular network.

Within a given microenvironment, the angiogenic response results from a balance between pro-angiogenic and anti-angiogenic factors, secreted both by tumor cells and components of the stroma; the prevalence of the former determines the “angiogenic switch”, resulting in the activation of angiogenesis followed by tumor outgrowth (Hanahan and Folkman, 1996. Cell 86(3):353-64).

Gene therapy based strategies of angiogenesis inhibition and especially the approach according to the present invention have several advantages compared with conventional modalities of administration of anti-angiogenic drugs. First of all, since effective suppression of pathological angiogenesis may eventually require chronic treatment, the gene therapy strategy according to the invention is useful to achieve selective delivery to affected tissues and prolonged expression of the therapeutic agents. Gene therapy in general also represents a method for circumventing the production problems of many recombinant proteins including their stability and solubility; adequate production of anti-angiogenic factors by recombinant engineering methods has been sometimes problematic (e.g. for angiostatin) and may limit their clinical application. Moreover gene transfer usage allows the correct folding of proteic agents and their stability in vivo since they are assembled in their physiologic environment. A particularly attractive feature of the inventive approach is the possibility of targeting gene delivery to selective tissues, namely tumor tissue, thus achieving localized gene expression and high regional drug concentrations without increasing the systemic levels of the therapeutic agents and thereby resulting in an improved therapeutic index.

Angiogenesis inhibitors are heterogeneous in origin and potency, and their growing list includes proteolysis products of larger molecules with a different function, such as angiostatin, endostatin and vasostatin, modulators of vascular endothelial growth factor activity, such as soluble FLT-1 (sFLT-1), and some cytokines/chemokines with marked anti-endothelial activity, such as IL-12, IFN-α, and CXCL10. The following table 8 (adapted from Persano et al., 2007. Mol. Aspects Med. 28(1):87-114. PMID: 17306361) summarizes the preferred angiogenesis inhibitors which may be used in the inventive approach. According to preferred embodiments in the context of the present invention angiogenesis inhibitors may be selected from any endogenous angiogenesis inhibitor selected from the group consisting of Angiopoietin-2; Angiostatin; Canstatin; CXCL10; CXCL4; CXCL9; Endostatin; FLK-1; IFNalpha; IFNB; IFNG; IL-12; PEX; PRL; SERPINB5; sFLT-1; sTie2; TIMP-1; TIMP-2; TIMP-3; TNF; TSP-1; TSP-2; Tumstatin; Vasostatin, preferably as disclosed in Table 7. Particularly preferred in this context are the RNA sequences encoding an angiogenesis inhibitor according to Table 7.

TABLE 7 Endogenous angiogenesis inhibitors RNA Optimized Protein Sequence RNA Sequence wild type Sequence Gene Name Protein Accession No. SEQ ID NO: SEQ ID NO: SEQ ID NO: IFNalpha UniProtKB: G9JKF1 3953 3954 3955, 3956, 3957, 3958, 3959, 3960 IFNalpha UniProtKB: P01562 3961 3962 3963, 3964, 3965, 3966, 3967, 3968 IFNalpha UniProtKB: P01563 3969 3970 3971, 3972, 3973, 3974, 3975, 3976 IFNalpha UniProtKB: P01566 3977 3978 3979, 3980, 3981, 3982, 3983, 3984 IFNalpha UniProtKB: P01567 3985 3986 3987, 3988, 3989, 3990, 3991, 3992 IFNalpha UniProtKB: P01568 3993 3994 3995, 3996, 3997, 3998, 3999, 4000 IFNalpha UniProtKB: P01569 4001 4002 4003, 4004, 4005, 4006, 4007, 4008 IFNalpha UniProtKB: P01570 4009 4010 4011, 4012, 4013, 4014, 4015, 4016 IFNalpha UniProtKB: P01571 4017 4018 4019, 4020, 4021, 4022, 4023, 4024 IFNalpha UniProtKB: P05013 4025 4026 4027, 4028, 4029, 4030, 4031, 4032 IFNalpha UniProtKB: P05014 4033 4034 4035, 4036, 4037, 4038, 4039, 4040 IFNalpha UniProtKB: P05015 4041 4042 4043, 4044, 4045, 4046, 4047, 4048 IFNalpha UniProtKB: P32881 4049 4050 4051, 4052, 4053, 4054, 4055, 4056 IFNalpha UniProtKB: Q14618 4057 4058 4059, 4060, 4061, 4062, 4063, 4064 IFNalpha UniProtKB: Q86UP4 4065 4066 4067, 4068, 4069, 4070, 4071, 4072 IFNB UniProtKB: P01574 4073 4074 4075, 4076, 4077, 4078, 4079, 4080 IFNB UniProtKB: Q15943 4081 4082 4083, 4084, 4085, 4086, 4087, 4088 IFNG UniProtKB: P01579 4089 4090 4091, 4092, 4093, 4094, 4095, 4096 IFNG UniProtKB: Q14609 4097 4098 4099, 4100, 4101, 4102, 4103, 4104 IFNG UniProtKB: Q14610 4105 4106 4107, 4108, 4109, 4110, 4111, 4112 IFNG UniProtKB: Q14611 4113 4114 4115, 4116, 4117, 4118, 4119, 4120 IFNG UniProtKB: Q14612 4121 4122 4123, 4124, 4125, 4126, 4127, 4128 IFNG UniProtKB: Q14613 4129 4130 4131, 4132, 4133, 4134, 4135, 4136 IFNG UniProtKB: Q14614 4137 4138 4139, 4140, 4141, 4142, 4143, 4144 IFNG UniProtKB: Q14615 4145 4146 4147, 4148, 4149, 4150, 4151, 4152 IFNG UniProtKB: Q8NHY9 4153 4154 4155, 4156, 4157, 4158, 4159, 4160 IL-12 UniProtKB: P29460 4193 4194 4195, 4196, 4197, 4198, 4199, 4200 CXCL10 UniProtKB: A0A024RDA4 5129 5130 5131, 5132, 5133, 5134, 5135, 5136 CXCL10 UniProtKB: P02778 5137 5138 5139, 5140, 5141, 5142, 5143, 5144 CXCL4 UniProtKB: P02776 5225 5226 5227, 5228, 5229, 5230, 5231, 5232 CXCL9 UniProtKB: L8E8X0 5273 5274 5275, 5276, 5277, 5278, 5279, 5280 CXCL9 UniProtKB: Q07325 5281 5282 5283, 5284, 5285, 5286, 5287, 5288 TNF UniProtKB: P01375 7369 7370 7371, 7372, 7373, 7374, 7375, 7376 TNF UniProtKB: Q5STB3 7377 7378 7379, 7380, 7381, 7382, 7383, 7384 Angiopoietin-2 UniProtKB: B2R6E3 7385 7386 7387, 7388, 7389, 7390, 7391, 7392 Angiopoietin-2 UniProtKB: O15123 7393 7394 7395, 7396, 7397, 7398, 7399, 7400 Angiostatin UniProtKB: A0A0F7G8J1 7401 7402 7403, 7404, 7405, 7406, 7407, 7408 Angiostatin UniProtKB: P00747 7409 7410 7411, 7412, 7413, 7414, 7415, 7416 Angiostatin UniProtKB: Q5TEH5 7417 7418 7419, 7420, 7421, 7422, 7423, 7424 Canstatin UniProtKB: P08572 7425 7426 7427, 7428, 7429, 7430, 7431, 7432 Endostatin Homo_sapiens 7433 7434 7435, 7436, 7437, 7438, 7439, 7440 FLK-1 UniProtKB: P35968 7441 7442 7443, 7444, 7445, 7446, 7447, 7448 PEX UniProtKB: P78562 7449 7450 7451, 7452, 7453, 7454, 7455, 7456 PRL UniProtKB: P01236 7457 7458 7459, 7460, 7461, 7462, 7463, 7464 SERPINB5 UniProtKB: P36952 7465 7466 7467, 7468, 7469, 7470, 7471, 7472 sFLT-1 UniProtKB: H9N1E7 7473 7474 7475, 7476, 7477, 7478, 7479, 7480 sFLT-1 UniProtKB: H9N1E8 7481 7482 7483, 7484, 7485, 7486, 7487, 7488 sFLT-1 UniProtKB: L7RSL3 7489 7490 7491, 7492, 7493, 7494, 7495, 7496 sFLT-1 UniProtKB: P17948 7497 7498 7499, 7500, 7501, 7502, 7503, 7504 sTie2 UniProtKB: B5A953 7505 7506 7507, 7508, 7509, 7510, 7511, 7512 TIMP-1 UniProtKB: P01033 7513 7514 7515, 7516, 7517, 7518, 7519, 7520 TIMP-2 UniProtKB: P16035 7521 7522 7523, 7524, 7525, 7526, 7527, 7528 TIMP-3 UniProtKB: P35625 7529 7530 7531, 7532, 7533, 7534, 7535, 7536 TSP-1 UniProtKB: P07996 7537 7538 7539, 7540, 7541, 7542, 7543, 7544 TSP-2 UniProtKB: P35442 7545 7546 7547, 7548, 7549, 7550, 7551, 7552 Tumstatin UniProtKB: Q01955 7553 7554 7555, 7556, 7557, 7558, 7559, 7560 Vasostatin UniProtKB: P10645 7561 7562 7563, 7564, 7565, 7566, 7567, 7568

According to the present invention, in a more preferred embodiment, the inventive composition comprises at least one RNA, preferably an mRNA comprising at least one coding region encoding at least one angiogenesis inhibitor or a fragment or variant thereof, wherein the at least one coding region comprises an RNA sequence being identical or at least 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the RNA sequences according to the SEQ ID Nos as disclosed in Table 7.

7. Heat Shock Proteins

In a further preferred embodiment of the inventive RNA containing composition the RNA codes for at least one heat shock protein (HSP) or a fragment or variant thereof. Preferably, the heat shock protein may be chosen from the following list: HSP27, HSP47 (serpin H1), HSP60, HSP70, HSC70, GRP78 (BiP), HSP90, HSP110, GRP94 (gp96), GRP170 (ORP150), PDI/PDIA, CRT/CALR.

As reviewed by Graner et al. (Graner M W, Lillehei K O, Katsanis E. Endoplasmic reticulum chaperones and their roles in the immunogenicity of cancer vaccines. Front Oncol. 2015 Jan. 6; 4:379. doi: 10.3389/fonc.2014.00379) heat shock proteins play essential cellular housekeeping functions and are indispensible during protein synthesis, folding and transport across intracellular membranes as well as protein degradation. HSPs belong to a multiprotein family of chaperons which consists of, but is not limited to, HSP27, HSP47 (serpin H1), HSP60, HSP70, HSC70, GRP78 (BiP), HSP90, HSP110, GRP94 (gp96), GRP170 (ORP150), PDI/PDIA, CRT/CALR. In addition to the intracellular functions as chaperons, HSPs have been shown to play an important extracellular role as simulators of the immune responses particularly in tumor settings. Various literature reports demonstrated that tumor-derived HSP-peptide complexes induce anti-tumor immune responses very efficiently. The molecular mechanism of these observations has been elucidated. HSPs as chaperons have the capacity to bind denatured peptides including the antigenic ones and those complexes are internalized by antigen presenting cells (APCs) which eventually leads to antigen presentation and induction of immunity. In addition to their chaperon function, HSPs have been shown to trigger danger signals in the tumor microenvironment and thus stimulate macrophages and dendritic cells (DCs) to produce proinflammatory cytokines and enhance the induced immune responses.

According to preferred embodiments in the context of the present invention heat shock proteins may be selected from any heat shock protein selected from the group consisting of calreticulin; GRP170_(ORP150); GRP78_(BiP); GRP94_(gp96); HSC70; HSP110; HSP27; HSP47_(serpin_H1); HSP60; HSP70; HSP90; PDI/PDIA, preferably as disclosed in Table 8. Particularly preferred in this context are the RNA sequences encoding a heat shock protein according to Table 8.

TABLE 8 Heat shock proteins RNA Optimized Protein Sequence RNA Sequence wild type Sequence Gene Name Protein Accession No. SEQ ID NO: SEQ ID NO: SEQ ID NO: calreticulin UniProtKB: B4DHR1 7569 7570 7571, 7572, 7573, 7574, 7575, 7576 calreticulin UniProtKB: B4E2Y9 7577 7578 7579, 7580, 7581, 7582, 7583, 7584 calreticulin UniProtKB: P27797 7585 7586 7587, 7588, 7589, 7590, 7591, 7592 calreticulin UniProtKB: Q96L12 7593 7594 7595, 7596, 7597, 7598, 7599, 7600 GRP170_(ORP150) UniProtKB: Q9Y4L1 7601 7602 7603, 7604, 7605, 7606, 7607, 7608 GRP78_(BiP) UniProtKB: P11021 7609 7610 7611, 7612, 7613, 7614, 7615, 7616 GRP94_(gp96) UniProtKB: P14625 7617 7618 7619, 7620, 7621, 7622, 7623, 7624 HSC70 UniProtKB: P11142 7625 7626 7627, 7628, 7629, 7630, 7631, 7632 HSP110 UniProtKB: Q92598 7633 7634 7635, 7636, 7637, 7638, 7639, 7640 HSP27 UniProtKB: P04792 7641 7642 7643, 7644, 7645, 7646, 7647, 7648 HSP47_(serpin_H1) UniProtKB: P50454 7649 7650 7651, 7652, 7653, 7654, 7655, 7656 HSP60 UniProtKB: A0A024R3X4 7657 7658 7659, 7660, 7661, 7662, 7663, 7664 HSP60 UniProtKB: B3GQS7 7665 7666 7667, 7668, 7669, 7670, 7671, 7672 HSP60 UniProtKB: P10809 7673 7674 7675, 7676, 7677, 7678, 7679, 7680 HSP60 UniProtKB: Q0VDF9 7681 7682 7683, 7684, 7685, 7686, 7687, 7688 HSP70 UniProtKB: P38646 7689 7690 7691, 7692, 7693, 7694, 7695, 7696 HSP90 UniProtKB: P07900 7697 7698 7699, 7700, 7701, 7702, 7703, 7704 HSP90 UniProtKB: P08238 7705 7706 7707, 7708, 7709, 7710, 7711, 7712 PDI/PDIA UniProtKB: P07237 7713 7714 7715, 7716, 7717, 7718, 7719, 7720 PDI/PDIA UniProtKB: Q6YPB00 7721 7722 7723, 7724, 7725, 7726, 7727, 7728 PDI/PDIA UniProtKB: Q71S60 7729 7730 7731, 7732, 7733, 7734, 7735, 7736

According to the present invention, in a more preferred embodiment, the inventive composition comprises at least one RNA, preferably an mRNA comprising at least one coding region encoding at least one heat shock protein or a fragment or variant thereof, wherein the at least one coding region comprises an RNA sequence being identical or at least 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the RNA sequences according to the SEQ ID Nos as disclosed in Table 8.

8. Tumor Antigens

In a further preferred embodiment of the inventive RNA containing composition the composition may contain RNA, preferably mRNA which codes for at least one tumor antigen or a fragment or variant thereof, which are used for vaccination to induce an adaptive immune response according to the invention.

In this context tumor antigens are particularly preferred to be encoded by RNA, preferably mRNA comprised in the inventive RNA composition. It is particularly preferred that the inventive RNA composition comprises at least one RNA encoding at least one tumor antigen or a fragment or variant thereof.

Tumor antigens are preferably located on the surface of the (tumor) cell. Tumor antigens may also be selected from proteins, which are overexpressed in tumor cells compared to a normal cell. Furthermore, tumor antigens also includes antigens expressed in cells which are (were) not themselves (or originally not themselves) degenerated but are associated with the supposed tumor. Antigens which are connected with tumor-supplying vessels or (re)formation thereof, in particular those antigens which are associated with neovascularization, e.g. growth factors, such as VEGF, bFGF etc., are also included herein. Antigens connected with a tumor furthermore include antigens from cells or tissues, typically embedding the tumor. Further, some substances (usually proteins or peptides) are expressed in patients suffering (knowingly or not-knowingly) from a cancer disease and they occur in increased concentrations in the body fluids of said patients. These substances are also referred to as “tumor antigens”, however they are not antigens in the stringent meaning of an immune response inducing substance. The class of tumor antigens can be divided further into tumor-specific antigens (TSAs) and tumor-associated-antigens (TAAs). TSAs can only be presented by tumor cells and never by normal “healthy” cells. They typically result from a tumor specific mutation. TAAs, which are more common, are usually presented by both tumor and healthy cells. These antigens are recognized and the antigen-presenting cell can be destroyed by cytotoxic T cells. Additionally, tumor antigens can also occur on the surface of the tumor in the form of, e.g., a mutated receptor. In this case, they can be recognized by antibodies.

Further, tumor associated antigens may be classified as tissue-specific antigens, also called melanocyte-specific antigens, cancer-testis antigens and tumor-specific antigens. Cancer-testis antigens are typically understood to be peptides or proteins of germ-line associated genes which may be activated in a wide variety of tumors. Human cancer-testis antigens may be further subdivided into antigens which are encoded on the X chromosome, so-called CT-X antigens, and those antigens which are not encoded on the X chromosome, the so-called (non-X CT antigens). Cancer-testis antigens which are encoded on the X-chromosome comprises, for example, the family of melanoma antigen genes, the so-called MAGE-family. The genes of the MAGE-family may be characterised by a shared MAGE homology domain (MHD). Each of these antigens, i.e. melanocyte-specific antigens, cancer-testis antigens and tumor-specific antigens, may elicit autologous cellular and humoral immune response. Accordingly, the tumor antigen encoded by the inventive nucleic acid sequence is preferably a melanocyte-specific antigen, a cancer-testis antigen or a tumor-specific antigens, preferably it may be a CT-X antigen, a non-X CT-antigens, a binding partner for a CT-X antigen or a binding partner for a non-X CT-antigen or a tumor-specific antigen, more preferably a CT-X antigen, a binding partner for a non-X CT-antigen or a tumor-specific antigen.

Particular preferred tumor antigens are selected from the list consisting of 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1, alpha-5-beta-1-integrin, alpha-5-beta-6-integrin, alpha-actinin-4/m, alpha-methylacyl-coenzyme A racemase, ART-4, ARTC1/m, B7H4, BAGE-1, BCL-2, bcr/abl, beta-catenin/m, BING-4, BRCA1/m, BRCA2/m, CA 15-3/CA 27-29, CA 19-9, CA72-4, CA125, calreticulin, CAMEL, CASP-8/m, cathepsin B, cathepsin L, CD19, CD20, CD22, CD25, CDE30, CD33, CD4, CD52, CD55, CD56, CD80, CDC27/m, CDK4/m, CDKN2A/m, CEA, CLCA2, CML28, CML66, COA-1/m, coactosin-like protein, collage XXIII, COX-2, CT-9/BRD6, Cten, cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-10, DAM-6, DEK-CAN, EFTUD2/m, EGFR, ELF2/m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, ETV6-AML1, EZH2, FGF-5, FN, Frau-1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE7b, GAGE-8, GDEP, GnT-V, gp100, GPC3, GPNMB/m, HAGE, HAST-2, hepsin, Her2/neu, HERV-K-MEL, HLA-A*0201-R17I, HLA-A11/m, HLA-A2/m, HNE, homeobox NKX3.1, HOM-TES-14/SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HSP70-2M, HST-2, hTERT, iCE, IGF-1R, IL-13Ra2, IL-2R, IL-5, immature laminin receptor, kallikrein-2, kallikrein-4, Ki67, KIAA0205, KIAA0205/m, KK-LC-1, K-Ras/m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, mammaglobin A, MART-1/melan-A, MART-2, MART-2/m, matrix protein 22, MC1R, M-CSF, ME1/m, mesothelin, MG50/PXDN, MMP11, MN/CA IX-antigen, MRP-3, MUC-1, MUC-2, MUM-1/m, MUM-2/m, MUM-3/m, myosin class I/m, NA88-A, N-acetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP/m, NFYC/m, NGEP, NMP22, NPM/ALK, N-Ras/m, NSE, NY-ESO-B, NY-ESO-1, OA1, OFA-iLRP, OGT, OGT/m, OS-9, OS-9/m, osteocalcin, osteopontin, p15, p190 minor bcr-abl, p53, p53/m, PAGE-4, PAI-1, PAI-2, PAP, PART-1, PATE, PDEF, Pim-1-Kinase, Pin-1, Pml/PARalpha, POTE, PRAME, PRDX5/m, prostein, proteinase-3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK/m, RAGE-1, RBAF600/m, RHAMM/CD168, RU1, RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC, SIRT2/m, Sp17, SSX-1, SSX-2/HOM-MEL-40, SSX-4, STAMP-1, STEAP-1, survivin, survivin-2B, SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1, TGFbeta, TGFbetaRII, TGM-4, TPI/m, TRAG-3, TRG, TRP-1, TRP-2/6b, TRP/INT2, TRP-p8, tyrosinase, UPA, VEGFR1, VEGFR-2/FLK-1, and WT1. Such tumor antigens preferably may be selected from the group consisting of p53, CA125, EGFR, Her2/neu, hTERT, PAP, MAGE-A1, MAGE-A3, Mesothelin, MUC-1, GP100, MART-1, Tyrosinase, PSA, PSCA, PSMA, STEAP-1, VEGF, VEGFR1, VEGFR2, Ras, CEA or WT1, and more preferably from PAP, MAGE-A3, WT1, and MUC-1. Such tumor antigens preferably may be selected from the group consisting of MAGE-A1 (e.g. MAGE-A1 according to accession number M77481), MAGE-A2, MAGE-A3, MAGE-A6 (e.g. MAGE-A6 according to accession number NM_005363), MAGE-C1, MAGE-C2, melan-A (e.g. melan-A according to accession number NM_005511), GP100 (e.g. GP100 according to accession number M77348), tyrosinase (e.g. tyrosinase according to accession number NM_000372), surviving (e.g. survivin according to accession number AF077350), CEA (e.g. CEA according to accession number NM_004363), Her-2/neu (e.g. Her-2/neu according to accession number M11730), WT1 (e.g. WT1 according to accession number NM_000378), PRAME (e.g. PRAME according to accession number NM_006115), EGFRI (epidermal growth factor receptor 1) (e.g. EGFRI (epidermal growth factor receptor 1) according to accession number AF288738), MUC1, mucin-1 (e.g. mucin-1 according to accession number NM_002456), SEC61G (e.g. SEC61G according to accession number NM_014302), hTERT (e.g. hTERT accession number NM_198253), 5T4 (e.g. 5T4 according to accession number NM_006670), TRP-2 (e.g. TRP-2 according to accession number NM_001922), STEAP1, PCA, PSA, PSMA, etc.

According to preferred embodiments in the context of the present invention tumor antigens may be selected from any tumor antigen selected from the group consisting of 1A01_HLA-A/m; 1A02; 5T4; ACRBP; AFP; AKAP4; alpha-actinin-_4/m; alpha-methylacyl-coenzyme_A_racemase; ANDR; ART-4; ARTC1/m; AURKB; B2MG; B3GN5; B4GN1; B7H4; BAGE-1; BASI; BCL-2; bcr/abl; beta-catenin/m; BING-4; BIRC7; BRCA1/m; BY55; calreticulin; CAMEL; CASPA; Caspase_8; cathepsin_B; cathepsin_L; CD1A; CD1B; CD1C; CD1D; CD1E; CD20; CD22; CD276; CD33; CD3E; CD3Z; CD4; CD44_Isoform_1; CD44_Isoform_6; CD52; CD55; CD56; CD80; CD86; CD8A; CDC27/m; CDE30; CDK4/m; CDKN2A/m; CEA; CEAM6; CH3L2; CLCA2; CML28; CML66; COA-1/m; coactosin-like_protein; collagen_XXIII; COX-2; CP1B1; CSAG2; CT-_9/BRD6; CT45A1; CT55; CTAG2_Isoform_LAGE-1A; CTAG2_Isoform_LAGE-1B; CTCFL; Cten; cyclin_B1; cyclin_D1; cyp-B; DAM-10; DEP1A; E7; EF1A2; EFTUD2/m; EGFR; EGLN3; ELF2/m; EMMPRIN; EpCam; EphA2; EphA3; ErbB3; ERBB4; ERG; ETV6; EWS; EZH2; FABP7; FCGR3A_Version_1; FCGR3A_Version_2; FGF5; FGFR2; fibronectin; FOS; FOXP3; FUT1; G250; GAGE-1; GAGE-2; GAGE-3; GAGE-4; GAGE-5; GAGE-6; GAGE7b; GAGE-8_(GAGE-2D); GASR; GnT-V; GPC3; GPNMB/m; GRM3; HAGE; hepsin; Her2/neu; HLA-A2/m; homeobox_NKX3.1; HOM-TES-85; HPG1; HS71A; HS71B; HST-2; hTERT; iCE; IF2B3; IL-10; IL-13Ra2; IL2-RA; IL2-RB; IL2-RG; IL-5; IMP3; ITA5; ITB1; ITB6; kallikrein-2; kallikrein-4; KI20A; KIAA0205; KIF2C; KK-LC-1; LDLR; LGMN; LIRB2; LY6K; MAGA5; MAGA8; MAGAB; MAGE-_B1; MAGE-_E1; MAGE-A1; MAGE-A10; MAGE-A12; MAGE-A2; MAGE-A3; MAGE-A4; MAGE-A6; MAGE-A9; MAGE-B10; MAGE-B16; MAGE-B17; MAGE-B2; MAGE-B3; MAGE-B4; MAGE-B5; MAGE-B6; MAGE-C1; MAGE-C2; MAGE-C3; MAGE-D1; MAGE-D2; MAGE-D4; MAGE-E1_(MAGE1); MAGE-E2; MAGE-F1; MAGE-H1; MAGEL2; mammaglobin_A; MART-1/melan-A; MART-2; MC1_R; M-CSF; mesothelin; MITF; MMP1_1; MMP7; MUC-1; MUM-1/m; MUM-2/m; MYO1A; MYO1B; MYO1C; MYO1D; MYO1E; MYO1F; MYO1G; MYO1H; NA17; NA88-A; Neo-PAP; NFYC/m; NGEP; N-myc; NPM; NRCAM; NSE; NUF2; NY-ESO-1; OA1; OGT; OS-9; osteocalcin; osteopontin; p53; PAGE-4; PAI-1; PAI-2; PAP; PATE; PAX3; PAX5; PD1L1; PDCD1; PDEF; PECA1; PGCB; PGFRB; Pim-1_-Kinase; Pin-1; PLAC1; PMEL; PML; POTE; POTEF; PRAME; PRDX5/m; PRM2; prostein; proteinase-3; PSA; PSB9; PSCA; PSGR; PSM; PTPRC; RAB8A; RAGE-1; RARA; RASH; RASK; RASN; RGS5; RHAMM/CD168; RHOC; RSSA; RU1; RU2; RUNX1; S-100; SAGE; SART-_1; SART-2; SART-3; SEPR; SERPINB5; SIA7F; SIA8A; SIAT9; SIRT2/m; SOX10; SP17; SPNXA; SPXN3; SSX-1; SSX-2; SSX3; SSX-4; ST1A1; STAG2; STAMP-1; STEAP-1; survivin; Survivin-2B; SYCP1; SYT-SSX-1; SYT-SSX-2; TARP; TCRg; TF2AA; TGFbeta1; TGFR2; TGM-4; TIE2; TKTL1; TPI/m; TRGV11; TRGV9; TRPC1; TRP-p8; TSG10; TSPY1; TVC_(TRGV3); TX101; tyrosinase; TYRP1; TYRP2; UPA; VEGFR1; WT1; XAGE1, preferably as disclosed in Table 9. Particularly preferred in this context are the RNA sequences encoding a tumor antigen according to Table 9.

TABLE 9 Tumor antigens RNA Optimized Protein Sequence RNA Sequence wild type Sequence Gene Name Protein Accession No. SEQ ID NO: SEQ ID NO: SEQ ID NO: 1A01_HLA-A/m UniProtKB: P30443 398 399 400, 401, 402, 403, 404 1A02 UniProtKB: P01892 405 406 407, 408, 409, 410, 411 5T4 UniProtKB: Q13641 412 413 414, 415, 416, 417, 418 ACRBP UniProtKB: Q8NEB7 419 420 421, 422, 423, 424, 425 AFP UniProtKB: P02771 426 427 428, 429, 430, 431, 432 AKAP4 UniProtKB: Q5JQC9 433 434 435, 436, 437, 438, 439 alpha-actinin-_4/m UniProtKB: B4DSX0 440 441 442, 443, 444, 445, 446 alpha-actinin-_4/m UniProtKB: B4E337 447 448 449, 450, 451, 452, 453 alpha-actinin-_4/m UniProtKB: O43707 454 455 456, 457, 458, 459, 460 alpha-methylacyl- UniProtKB: A0A024RE16 461 462 463, 464, 465, 466, 467 coenzyme_A_racemase alpha-methylacyl- UniProtKB: A8KAC3 468 469 470, 471, 472, 473, 474 coenzyme_A_racemase ANDR UniProtKB: P10275 475 476 477, 478, 479, 480, 481 ART-4 UniProtKB: Q9ULX3 482 483 484, 485, 486, 487, 488 ARTC1/m UniProtKB: P52961 489 490 491, 492, 493, 494, 495 AURKB UniProtKB: Q96GD4 496 497 498, 499, 500, 501, 502 B2MG UniProtKB: P61769 503 504 505, 506, 507, 508, 509 B3GN5 UniProtKB: Q9BYG0 510 511 512, 513, 514, 515, 516 B4GN1 UniProtKB: Q00973 517 518 519, 520, 521, 522, 523 B7H4 UniProtKB: Q7Z7D3 524 525 526, 527, 528, 529, 530 BAGE-1 UniProtKB: Q13072 531 532 533, 534, 535, 536, 537 BASI UniProtKB: P35613 538 539 540, 541, 542, 543, 544 BCL-2 UniProtKB: A9QXG9 545 546 547, 548, 549, 550, 551 bcr/abl UniProtKB: A9UEZ4 552 553 554, 555, 556, 557, 558 bcr/abl UniProtKB: A9UEZ7 559 560 561, 562, 563, 564, 565 bcr/abl UniProtKB: A9UEZ8 566 567 568, 569, 570, 571, 572 bcr/abl UniProtKB: A9UEZ9 573 574 575, 576, 577, 578, 579 bcr/abl UniProtKB: A9UF00 580 581 582, 583, 584, 585, 586 bcr/abl UniProtKB: A9UF01 587 588 589, 590, 591, 592, 593 bcr/abl UniProtKB: A9UF03 594 595 596, 597, 598, 599, 600 bcr/abl UniProtKB: A9UF04 601 602 603, 604, 605, 606, 607 bcr/abl UniProtKB: A9UF05 608 609 610, 611, 612, 613, 614 bcr/abl UniProtKB: A9UF06 615 616 617, 618, 619, 620, 621 bcr/abl UniProtKB: A9UF08 622 623 624, 625, 626, 627, 628 beta-catenin/m UniProtKB: P35222 629 630 631, 632, 633, 634, 635 beta-catenin/m UniProtKB: Q8WYA6 636 637 638, 639, 640, 641, 642 BING-4 UniProtKB: O15213 643 644 645, 646, 647, 648, 649 BIRC7 UniProtKB: Q96CA5 650 651 652, 653, 654, 655, 656 BRCA1/m UniProtKB: A0A024R1V0 657 658 659, 660, 661, 662, 663 BRCA1/m UniProtKB: A0A024R1V7 664 665 666, 667, 668, 669, 670 BRCA1/m UniProtKB: A0A024R1Z8 671 672 673, 674, 675, 676, 677 BRCA1/m UniProtKB: A0A068BFX7 678 679 680, 681, 682, 683, 684 BRCA1/m UniProtKB: C6YB45 685 686 687, 688, 689, 690, 691 BRCA1/m UniProtKB: C6YB47 692 693 694, 695, 696, 697, 698 BRCA1/m UniProtKB: G3XAC3 699 700 701, 702, 703, 704, 705 BY55 UniProtKB: O95971 706 707 708, 709, 710, 711, 712 CAMEL UniProtKB: O95987 713 714 715, 716, 717, 718, 719 CASPA UniProtKB: Q92851-4 720 721 722, 723, 724, 725, 726 cathepsin_B UniProtKB: A0A024R374 727 728 729, 730, 731, 732, 733 cathepsin_B UniProtKB: P07858 734 735 736, 737, 738, 739, 740 cathepsin_L UniProtKB: A0A024R276 741 742 743, 744, 745, 746, 747 cathepsin_L UniProtKB: P07711 748 749 750, 751, 752, 753, 754 cathepsin_L UniProtKB: Q9HBQ7 755 756 757, 758, 759, 760, 761 CD1A UniProtKB: P06126 762 763 764, 765, 766, 767, 768 CD1B UniProtKB: P29016 769 770 771, 772, 773, 774, 775 CD1C UniProtKB: P29017 776 777 778, 779, 780, 781, 782 CD1D UniProtKB: P15813 783 784 785, 786, 787, 788, 789 CD1E UniProtKB: P15812 790 791 792, 793, 794, 795, 796 CD20 UniProtKB: P11836 797 798 799, 800, 801, 802, 803 CD22 UniProtKB: O60926 804 805 806, 807, 808, 809, 810 CD22 UniProtKB: P20273 811 812 813, 814, 815, 816, 817 CD22 UniProtKB: Q0EAF5 818 819 820, 821, 822, 823, 824 CD276 UniProtKB: Q5ZPR3 825 826 827, 828, 829, 830, 831 CD33 UniProtKB: B4DF51 832 833 834, 835, 836, 837, 838 CD33 UniProtKB: P20138 839 840 841, 842, 843, 844, 845 CD33 UniProtKB: Q546G0 846 847 848, 849, 850, 851, 852 CD3E UniProtKB: P07766 853 854 855, 856, 857, 858, 859 CD3Z UniProtKB: P20963 860 861 862, 863, 864, 865, 866 CD44_Isoform_1 UniProtKB: P16070 867 868 869, 870, 871, 872, 873 CD44_Isoform_6 UniProtKB: P16070-6 874 875 876, 877, 878, 879, 880 CD4 UniProtKB: P01730 881 882 883, 884, 885, 886, 887 CD52 UniProtKB: P31358 888 889 890, 891, 892, 893, 894 CD52 UniProtKB: Q6IBD0 895 896 897, 898, 899, 900, 901 CD52 UniProtKB: V9HWN9 902 903 904, 905, 906, 907, 908 CD55 UniProtKB: B1AP15 909 910 911, 912, 913, 914, 915 CD55 UniProtKB: D3DT85 916 917 918, 919, 920, 921, 922 CD55 UniProtKB: D3DT86 923 924 925, 926, 927, 928, 929 CD55 UniProtKB: P08174 930 931 932, 933, 934, 935, 936 CD56 UniProtKB: P13591 937 938 939, 940, 941, 942, 943 CD80 UniProtKB: A0N0P2 944 945 946, 947, 948, 949, 950 CD80 UniProtKB: P33681 951 952 953, 954, 955, 956, 957 CD86 UniProtKB: P42081 958 959 960, 961, 962, 963, 964 CD8A UniProtKB: P01732 965 966 967, 968, 969, 970, 971 CDC27/m UniProtKB: G5EA36 972 973 974, 975, 976, 977, 978 CDC27/m UniProtKB: P30260 979 980 981, 982, 983, 984, 985 CDE30 UniProtKB: P28908 986 987 988, 989, 990, 991, 992 CDK4/m UniProtKB: A0A024RBB6 993 994 995, 996, 997, 998, 999 CDK4/m UniProtKB: P11802 1000 1001 1002, 1003, 1004, 1005, 1006 CDK4/m UniProtKB: Q6LC83 1007 1008 1009, 1010, 1011, 1012, 1013 CDK4/m UniProtKB: Q96BE9 1014 1015 1016, 1017, 1018, 1019, 1020 CDKN2A/m UniProtKB: D1LYX3 1021 1022 1023, 1024, 1025, 1026, 1027 CDKN2A/m UniProtKB: G3XAG3 1028 1029 1030, 1031, 1032, 1033, 1034 CDKN2A/m UniProtKB: K7PML8 1035 1036 1037, 1038, 1039, 1040, 1041 CDKN2A/m UniProtKB: L8E941 1042 1043 1044, 1045, 1046, 1047, 1048 CDKN2A/m UniProtKB: Q8N726 1049 1050 1051, 1052, 1053, 1054, 1055 CEA RefSeq: NP_004354 1056 1057 1058, 1059, 1060, 1061, 1062 CEAM6 UniProtKB: P40199 1063 1064 1065, 1066, 1067, 1068, 1069 CH3L2 UniProtKB: Q15782 1070 1071 1072, 1073, 1074, 1075, 1076 CLCA2 UniProtKB: Q9UQC9 1077 1078 1079, 1080, 1081, 1082, 1083 CML28 UniProtKB: Q9NQT4 1084 1085 1086, 1087, 1088, 1089, 1090 CML66 UniProtKB: Q96RS6 1091 1092 1093, 1094, 1095, 1096, 1097 COA-1/m UniProtKB: Q5T124 1098 1099 1100, 1101, 1102, 1103, 1104 coactosin-like_protein UniProtKB: Q14019 1105 1106 1107, 1108, 1109, 1110, 1111 collagen_XXIII UniProtKB: L8EAS4 1112 1113 1114, 1115, 1116, 1117, 1118 collagen_XXIII UniProtKB: Q86Y22 1119 1120 1121, 1122, 1123, 1124, 1125 COX-2 UniProtKB: Q6ZYK7 1126 1127 1128, 1129, 1130, 1131, 1132 CP1B1 UniProtKB: Q16678 1133 1134 1135, 1136, 1137, 1138, 1139 CSAG2 UniProtKB: Q9Y5P2-2 1140 1141 1142, 1143, 1144, 1145, 1146 CSAG2 UniProtKB: Q9Y5P2 1147 1148 1149, 1150, 1151, 1152, 1153 CT45A1 UniProtKB: Q5HYN5 1154 1155 1156, 1157, 1158, 1159, 1160 CT55 UniProtKB: Q8WUE5 1161 1162 1163, 1164, 1165, 1166, 1167 CT-_9/BRD6 UniProtKB: Q58F21 1168 1169 1170, 1171, 1172, 1173, 1174 CTAG2_Isoform_LAGE- UniProtKB: O75638-2 1175 1176 1177, 1178, 1179, 1A 1180, 1181 CTAG2_Isoform_LAGE- UniProtKB: O75638 1182 1183 1184, 1185, 1186, 1B 1187, 1188 CTCFL UniProtKB: Q8NI51 1189 1190 1191, 1192, 1193, 1194, 1195 Cten UniProtKB: Q8IZW8 1196 1197 1198, 1199, 1200, 1201, 1202 cyclin_B1 UniProtKB: P14635 1203 1204 1205, 1206, 1207, 1208, 1209 cyclin_D1 UniProtKB: P24385 1210 1211 1212, 1213, 1214, 1215, 1216 cyp-B UniProtKB: P23284 1217 1218 1219, 1220, 1221, 1222, 1223 DAM-10 UniProtKB: P43366 1224 1225 1226, 1227, 1228, 1229, 1230 DEP1A UniProtKB: Q5TB30 1231 1232 1233, 1234, 1235, 1236, 1237 E7 UniProtKB: P03129 1238 1239 1240, 1241, 1242, 1243, 1244 E7 UniProtKB: P06788 1245 1246 1247, 1248, 1249, 1250, 1251 E7 UniProtKB: P17387 1252 1253 1254, 1255, 1256, 1257, 1258 E7 UniProtKB: P06429 1259 1260 1261, 1262, 1263, 1264, 1265 E7 UniProtKB: P27230 1266 1267 1268, 1269, 1270, 1271, 1272 E7 UniProtKB: P24837 1273 1274 1275, 1276, 1277, 1278, 1279 E7 UniProtKB: P21736 1280 1281 1282, 1283, 1284, 1285, 1286 E7 UniProtKB: P26558 1287 1288 1289, 1290, 1291, 1292, 1293 E7 UniProtKB: P36831 1294 1295 1296, 1297, 1298, 1299, 1300 E7 UniProtKB: P36833 1301 1302 1303, 1304, 1305, 1306, 1307 E7 UniProtKB: Q9QCZ1 1308 1309 1310, 1311, 1312, 1313, 1314 E7 UniProtKB: Q81965 1315 1316 1317, 1318, 1319, 1320, 1321 E7 UniProtKB: Q80956 1322 1323 1324, 1325, 1326, 1327, 1328 EF1A2 UniProtKB: Q05639 1329 1330 1331, 1332, 1333, 1334, 1335 EFTUD2/m UniProtKB: Q15029 1336 1337 1338, 1339, 1340, 1341, 1342 EGFR UniProtKB: A0A0B4J1Y5 1343 1344 1345, 1346, 1347, 1348, 1349 EGFR UniProtKB: E7BSV0 1350 1351 1352, 1353, 1354, 1355, 1356 EGFR UniProtKB: L0R6G1 1357 1358 1359, 1360, 1361, 1362, 1363 EGFR UniProtKB: P00533-2 1364 1365 1366, 1367, 1368, 1369, 1370 EGFR UniProtKB: P00533 1371 1372 1373, 1374, 1375, 1376, 1377 EGFR UniProtKB: Q147T7 1378 1379 1380, 1381, 1382, 1383, 1384 EGFR UniProtKB: Q504U8 1385 1386 1387, 1388, 1389, 1390, 1391 EGFR UniProtKB: Q8NDU8 1392 1393 1394, 1395, 1396, 1397, 1398 EGLN3 UniProtKB: Q9H6Z9 1399 1400 1401, 1402, 1403, 1404, 1405 ELF2/m UniProtKB: B7Z720 1406 1407 1408, 1409, 1410, 1411, 1412 EMMPRIN UniProtKB: Q54A51 1413 1414 1415, 1416, 1417, 1418, 1419 EpCam UniProtKB: P16422 1420 1421 1422, 1423, 1424, 1425, 1426 EphA2 UniProtKB: P29317 1427 1428 1429, 1430, 1431, 1432, 1433 EphA3 UniProtKB: P29320 1434 1435 1436, 1437, 1438, 1439, 1440 EphA3 UniProtKB: Q6P4R6 1441 1442 1443, 1444, 1445, 1446, 1447 ErbB3 UniProtKB: B3KWG5 1448 1449 1450, 1451, 1452, 1453, 1454 ErbB3 UniProtKB: B4DGQ7 1455 1456 1457, 1458, 1459, 1460, 1461 ERBB4 UniProtKB: Q15303 1462 1463 1464, 1465, 1466, 1467, 1468 ERG UniProtKB: P11308 1469 1470 1471, 1472, 1473, 1474, 1475 ETV6 UniProtKB: P41212 1476 1477 1478, 1479, 1480, 1481, 1482 EWS UniProtKB: Q01844 1483 1484 1485, 1486, 1487, 1488, 1489 EZH2 UniProtKB: F2YMM1 1490 1491 1492, 1493, 1494, 1495, 1496 EZH2 UniProtKB: G3XAL2 1497 1498 1499, 1500, 1501, 1502, 1503 EZH2 UniProtKB: L0R855 1504 1505 1506, 1507, 1508, 1509, 1510 EZH2 UniProtKB: Q15910 1511 1512 1513, 1514, 1515, 1516, 1517 EZH2 UniProtKB: S4S3R8 1518 1519 1520, 1521, 1522, 1523, 1524 FABP7 UniProtKB: O15540 1525 1526 1527, 1528, 1529, 1530, 1531 FCGR3A_Version_1 UniProtKB: P08637 1532 1533 1534, 1535, 1536, 1537, 1538 FCGR3A_Version_2 CCDS: CCDS1232.1 1539 1540 1541, 1542, 1543, 1544, 1545 FGF5 UniProtKB: P12034 1546 1547 1548, 1549, 1550, 1551, 1552 FGF5 UniProtKB: Q60518 1553 1554 1555, 1556, 1557, 1558, 1559 FGFR2 UniProtKB: P21802 1560 1561 1562, 1563, 1564, 1565, 1566 fibronectin UniProtKB: A0A024R5I6 1567 1568 1569, 1570, 1571, 1572, 1573 fibronectin UniProtKB: A0A024RB01 1574 1575 1576, 1577, 1578, 1579, 1580 fibronectin UniProtKB: A0A024RDT9 1581 1582 1583, 1584, 1585, 1586, 1587 fibronectin UniProtKB: A0A024RDV5 1588 1589 1590, 1591, 1592, 1593, 1594 fibronectin UniProtKB: A6NH44 1595 1596 1597, 1598, 1599, 1600, 1601 fibronectin UniProtKB: A8K6A5 1602 1603 1604, 1605, 1606, 1607, 1608 fibronectin UniProtKB: B2R627 1609 1610 1611, 1612, 1613, 1614, 1615 fibronectin UniProtKB: B3KXM5 1616 1617 1618, 1619, 1620, 1621, 1622 fibronectin UniProtKB: B4DIC5 1623 1624 1625, 1626, 1627, 1628, 1629 fibronectin UniProtKB: B4DN21 1630 1631 1632, 1633, 1634, 1635, 1636 fibronectin UniProtKB: B4DS98 1637 1638 1639, 1640, 1641, 1642, 1643 fibronectin UniProtKB: B4DTH2 1644 1645 1646, 1647, 1648, 1649, 1650 fibronectin UniProtKB: B4DTK1 1651 1652 1653, 1654, 1655, 1656, 1657 fibronectin UniProtKB: B4DU16 1658 1659 1660, 1661, 1662, 1663, 1664 fibronectin UniProtKB: B7Z3W5 1665 1666 1667, 1668, 1669, 1670, 1671 fibronectin UniProtKB: B7Z939 1672 1673 1674, 1675, 1676, 1677, 1678 fibronectin UniProtKB: G5E9X3 1679 1680 1681, 1682, 1683, 1684, 1685 fibronectin UniProtKB: Q9H382 1686 1687 1688, 1689, 1690, 1691, 1692 FOS UniProtKB: P01100 1693 1694 1695, 1696, 1697, 1698, 1699 FOXP3 UniProtKB: Q9BZS1 1700 1701 1702, 1703, 1704, 1705, 1706 FUT1 UniProtKB: P19526 1707 1708 1709, 1710, 1711, 1712, 1713 G250 UniProtKB: Q16790 1714 1715 1716, 1717, 1718, 1719, 1720 GAGE-1 Genbank: AAA82744 1721 1722 1723, 1724, 1725, 1726, 1727 GAGE-2 UniProtKB: Q6NT46 1728 1729 1730, 1731, 1732, 1733, 1734 GAGE-3 UniProtKB: Q13067 1735 1736 1737, 1738, 1739, 1740, 1741 GAGE-4 UniProtKB: Q13068 1742 1743 1744, 1745, 1746, 1747, 1748 GAGE-5 UniProtKB: Q13069 1749 1750 1751, 1752, 1753, 1754, 1755 GAGE-6 UniProtKB: Q13070 1756 1757 1758, 1759, 1760, 1761, 1762 GAGE7b UniProtKB: O76087 1763 1764 1765, 1766, 1767, 1768, 1769 GAGE-8_(GAGE-2D) UniProtKB: Q9UEU5 1770 1771 1772, 1773, 1774, 1775, 1776 GASR UniProtKB: P32239 1777 1778 1779, 1780, 1781, 1782, 1783 GnT-V UniProtKB: Q09328 1784 1785 1786, 1787, 1788, 1789, 1790 GPC3 UniProtKB: I6QJG3 1791 1792 1793, 1794, 1795, 1796, 1797 GPC3 UniProtKB: P51654 1798 1799 1800, 1801, 1802, 1803, 1804 GPC3 UniProtKB: Q8IYG2 1805 1806 1807, 1808, 1809, 1810, 1811 GPNMB/m UniProtKB: A0A024RA55 1812 1813 1814, 1815, 1816, 1817, 1818 GPNMB/m UniProtKB: Q14956 1819 1820 1821, 1822, 1823, 1824, 1825 GPNMB/m UniProtKB: Q8IXJ5 1826 1827 1828, 1829, 1830, 1831, 1832 GPNMB/m UniProtKB: Q96F58 1833 1834 1835, 1836, 1837, 1838, 1839 GRM3 UniProtKB: Q14832 1840 1841 1842, 1843, 1844, 1845, 1846 HAGE UniProtKB: Q9NXZ2 1847 1848 1849, 1850, 1851, 1852, 1853 hepsin UniProtKB: B2ZDQ2 1854 1855 1856, 1857, 1858, 1859, 1860 hepsin UniProtKB: P05981 1861 1862 1863, 1864, 1865, 1866, 1867 Her2/neu UniProtKB: B4DTR1 1868 1869 1870, 1871, 1872, 1873, 1874 Her2/neu UniProtKB: L8E8G2 1875 1876 1877, 1878, 1879, 1880, 1881 Her2/neu UniProtKB: P04626 1882 1883 1884, 1885, 1886, 1887, 1888 Her2/neu UniProtKB: Q9UK79 1889 1890 1891, 1892, 1893, 1894, 1895 HLA-A2/m UniProtKB: Q95387 1896 1897 1898, 1899, 1900, 1901, 1902 HLA-A2/m UniProtKB: Q9MYF8 1903 1904 1905, 1906, 1907, 1908, 1909 homeobox_NKX3.1 UniProtKB: Q99801 1910 1911 1912, 1913, 1914, 1915, 1916 HOM-TES-85 UniProtKB: B2RBQ6 1917 1918 1919, 1920, 1921, 1922, 1923 HOM-TES-85 UniProtKB: Q9P127 1924 1925 1926, 1927, 1928, 1929, 1930 HPG1 Pubmed: 12543784 1931 1932 1933, 1934, 1935, 1936, 1937 HS71A UniProtKB: P0DMV8 1938 1939 1940, 1941, 1942, 1943, 1944 HS71B UniProtKB: P0DMV9 1945 1946 1947, 1948, 1949, 1950, 1951 HST-2 UniProtKB: P10767 1952 1953 1954, 1955, 1956, 1957, 1958 hTERT UniProtKB: O94807 1959 1960 1961, 1962, 1963, 1964, 1965 iCE UniProtKB: O00748 1966 1967 1968, 1969, 1970, 1971, 1972 IF2B3 UniProtKB: O00425 1973 1974 1975, 1976, 1977, 1978, 1979 IL-13Ra2 UniProtKB: Q14627 1980 1981 1982, 1983, 1984, 1985, 1986 IL2-RA UniProtKB: P01589 1987 1988 1989, 1990, 1991, 1992, 1993 IL2-RB UniProtKB: P14784 1994 1995 1996, 1997, 1998, 1999, 2000 IL2-RG UniProtKB: P31785 2001 2002 2003, 2004, 2005, 2006, 2007 IMP3 UniProtKB: Q9NV31 2008 2009 2010, 2011, 2012, 2013, 2014 ITA5 UniProtKB: P08648 2015 2016 2017, 2018, 2019, 2020, 2021 ITB1 UniProtKB: P05556 2022 2023 2024, 2025, 2026, 2027, 2028 ITB6 UniProtKB: P18564 2029 2030 2031, 2032, 2033, 2034, 2035 kallikrein-2 UniProtKB: A0A024R4J4 2036 2037 2038, 2039, 2040, 2041, 2042 kallikrein-2 UniProtKB: A0A024R4N3 2043 2044 2045, 2046, 2047, 2048, 2049 kallikrein-2 UniProtKB: B0AZU9 2050 2051 2052, 2053, 2054, 2055, 2056 kallikrein-2 UniProtKB: B4DU77 2057 2058 2059, 2060, 2061, 2062, 2063 kallikrein-2 UniProtKB: P20151 2064 2065 2066, 2067, 2068, 2069, 2070 kallikrein-2 UniProtKB: Q6T774 2071 2072 2073, 2074, 2075, 2076, 2077 kallikrein-2 UniProtKB: Q6T775 2078 2079 2080, 2081, 2082, 2083, 2084 kallikrein-4 UniProtKB: A0A0C4DFQ5 2085 2086 2087, 2088, 2089, 2090, 2091 kallikrein-4 UniProtKB: Q5BQA0 2092 2093 2094, 2095, 2096, 2097, 2098 kallikrein-4 UniProtKB: Q96PT0 2099 2100 2101, 2102, 2103, 2104, 2105 kallikrein-4 UniProtKB: Q96PT1 2106 2107 2108, 2109, 2110, 2111, 2112 kallikrein-4 UniProtKB: Q9Y5K2 2113 2114 2115, 2116, 2117, 2118, 2119 KI20A UniProtKB: O95235 2120 2121 2122, 2123, 2124, 2125, 2126 KIAA0205 UniProtKB: Q92604 2127 2128 2129, 2130, 2131, 2132, 2133 KIF2C UniProtKB: Q99661 2134 2135 2136, 2137, 2138, 2139, 2140 KK-LC-1 UniProtKB: Q5H943 2141 2142 2143, 2144, 2145, 2146, 2147 LDLR UniProtKB: P01130 2148 2149 2150, 2151, 2152, 2153, 2154 LGMN UniProtKB: Q99538 2155 2156 2157, 2158, 2159, 2160, 2161 LIRB2 UniProtKB: Q8N423 2162 2163 2164, 2165, 2166, 2167, 2168 LY6K UniProtKB: Q17RY6 2169 2170 2171, 2172, 2173, 2174, 2175 MAGA5 UniProtKB: P43359 2176 2177 2178, 2179, 2180, 2181, 2182 MAGA8 UniProtKB: P43361 2183 2184 2185, 2186, 2187, 2188, 2189 MAGAB UniProtKB: P43364 2190 2191 2192, 2193, 2194, 2195, 2196 MAGE-A10 UniProtKB: A0A024RC14 2197 2198 2199, 2200, 2201, 2202, 2203 MAGE-A12 UniProtKB: P43365 2204 2205 2206, 2207, 2208, 2209, 2210 MAGE-A1 UniProtKB: P43355 2211 2212 2213, 2214, 2215, 2216, 2217 MAGE-A2 UniProtKB: P43356 2218 2219 2220, 2221, 2222, 2223, 2224 MAGE-A3 UniProtKB: P43357 2225 2226 2227, 2228, 2229, 2230, 2231 MAGE-A4 UniProtKB: A0A024RC12 2232 2233 2234, 2235, 2236, 2237, 2238 MAGE-A4 UniProtKB: P43358 2239 2240 2241, 2242, 2243, 2244, 2245 MAGE-A4 UniProtKB: Q1RN33 2246 2247 2248, 2249, 2250, 2251, 2252 MAGE-A6 UniProtKB: A8K072 2253 2254 2255, 2256, 2257, 2258, 2259 MAGE-A6 UniProtKB: P43360 2260 2261 2262, 2263, 2264, 2265, 2266 MAGE-A6 UniProtKB: Q6FHI5 2267 2268 2269, 2270, 2271, 2272, 2273 MAGE-A9 UniProtKB: P43362 2274 2275 2276, 2277, 2278, 2279, 2280 MAGE-B10 UniProtKB: Q96LZ2 2281 2282 2283, 2284, 2285, 2286, 2287 MAGE-B16 UniProtKB: A2A368 2288 2289 2290, 2291, 2292, 2293, 2294 MAGE-B17 UniProtKB: A8MXT2 2295 2296 2297, 2298, 2299, 2300, 2301 MAGE-_B1 UniProtKB: Q96TG1 2302 2303 2304, 2305, 2306, 2307, 2308 MAGE-B2 UniProtKB: O15479 2309 2310 2311, 2312, 2313, 2314, 2315 MAGE-B3 UniProtKB: O15480 2316 2317 2318, 2319, 2320, 2321, 2322 MAGE-B4 UniProtKB: O15481 2323 2324 2325, 2326, 2327, 2328, 2329 MAGE-B5 UniProtKB: Q9BZ81 2330 2331 2332, 2333, 2334, 2335, 2336 MAGE-B6 UniProtKB: Q8N7X4 2337 2338 2339, 2340, 2341, 2342, 2343 MAGE-C1 UniProtKB: O60732 2344 2345 2346, 2347, 2348, 2349, 2350 MAGE-C2 UniProtKB: Q9UBF1 2351 2352 2353, 2354, 2355, 2356, 2357 MAGE-C3 UniProtKB: Q8TD91 2358 2359 2360, 2361, 2362, 2363, 2364 MAGE-D1 UniProtKB: Q9Y5V3 2365 2366 2367, 2368, 2369, 2370, 2371 MAGE-D2 UniProtKB: Q9UNF1 2372 2373 2374, 2375, 2376, 2377, 2378 MAGE-D4 UniProtKB: Q96JG8 2379 2380 2381, 2382, 2383, 2384, 2385 MAGE-_E1 UniProtKB: Q6IAI7 2386 2387 2388, 2389, 2390, 2391, 2392 MAGE-E1_(MAGE1) UniProtKB: Q9HCI5 2393 2394 2395, 2396, 2397, 2398, 2399 MAGE-E2 UniProtKB: Q8TD90 2400 2401 2402, 2403, 2404, 2405, 2406 MAGE-F1 UniProtKB: Q9HAY2 2407 2408 2409, 2410, 2411, 2412, 2413 MAGE-H1 UniProtKB: Q9H213 2414 2415 2416, 2417, 2418, 2419, 2420 MAGEL2 UniProtKB: Q9UJ55 2421 2422 2423, 2424, 2425, 2426, 2427 mammaglobin_A UniProtKB: Q13296 2428 2429 2430, 2431, 2432, 2433, 2434 mammaglobin_A UniProtKB: Q6NX70 2435 2436 2437, 2438, 2439, 2440, 2441 MART-1/melan-A UniProtKB: Q16655 2442 2443 2444, 2445, 2446, 2447, 2448 MART-2 UniProtKB: Q5VTY9 2449 2450 2451, 2452, 2453, 2454, 2455 MC1_R UniProtKB: Q01726 2456 2457 2458, 2459, 2460, 2461, 2462 MC1_R UniProtKB: Q1JUL4 2463 2464 2465, 2466, 2467, 2468, 2469 MC1_R UniProtKB: Q1JUL6 2470 2471 2472, 2473, 2474, 2475, 2476 MC1_R UniProtKB: Q1JUL8 2477 2478 2479, 2480, 2481, 2482, 2483 MC1_R UniProtKB: Q1JUL9 2484 2485 2486, 2487, 2488, 2489, 2490 MC1_R UniProtKB: Q1JUM0 2491 2492 2493, 2494, 2495, 2496, 2497 MC1_R UniProtKB: Q1JUM2 2498 2499 2500, 2501, 2502, 2503, 2504 MC1_R UniProtKB: Q1JUM3 2505 2506 2507, 2508, 2509, 2510, 2511 MC1_R UniProtKB: Q1JUM4 2512 2513 2514, 2515, 2516, 2517, 2518 MC1_R UniProtKB: Q1JUM5 2519 2520 2521, 2522, 2523, 2524, 2525 MC1_R UniProtKB: Q6UR92 2526 2527 2528, 2529, 2530, 2531, 2532 MC1_R UniProtKB: Q6UR94 2533 2534 2535, 2536, 2537, 2538, 2539 MC1_R UniProtKB: Q6UR95 2540 2541 2542, 2543, 2544, 2545, 2546 MC1_R UniProtKB: Q6UR96 2547 2548 2549, 2550, 2551, 2552, 2553 MC1_R UniProtKB: Q6UR97 2554 2555 2556, 2557, 2558, 2559, 2560 MC1_R UniProtKB: Q6UR98 2561 2562 2563, 2564, 2565, 2566, 2567 MC1_R UniProtKB: Q6UR99 2568 2569 2570, 2571, 2572, 2573, 2574 MC1_R UniProtKB: Q6URA0 2575 2576 2577, 2578, 2579, 2580, 2581 MC1_R UniProtKB: Q86YW1 2582 2583 2584, 2585, 2586, 2587, 2588 MC1_R UniProtKB: V9Q5S2 2589 2590 2591, 2592, 2593, 2594, 2595 MC1_R UniProtKB: V9Q671 2596 2597 2598, 2599, 2600, 2601, 2602 MC1_R UniProtKB: V9Q783 2603 2604 2605, 2606, 2607, 2608, 2609 MC1_R UniProtKB: V9Q7F1 2610 2611 2612, 2613, 2614, 2615, 2616 MC1_R UniProtKB: V9Q8N1 2617 2618 2619, 2620, 2621, 2622, 2623 MC1_R UniProtKB: V9Q977 2624 2625 2626, 2627, 2628, 2629, 2630 MC1_R UniProtKB: V9Q9P5 2631 2632 2633, 2634, 2635, 2636, 2637 MC1_R UniProtKB: V9Q9R8 2638 2639 2640, 2641, 2642, 2643, 2644 MC1_R UniProtKB: V9QAE0 2645 2646 2647, 2648, 2649, 2650, 2651 MC1_R UniProtKB: V9QAR2 2652 2653 2654, 2655, 2656, 2657, 2658 MC1_R UniProtKB: V9QAW3 2659 2660 2661, 2662, 2663, 2664, 2665 MC1_R UniProtKB: V9QB02 2666 2667 2668, 2669, 2670, 2671, 2672 MC1_R UniProtKB: V9QB58 2673 2674 2675, 2676, 2677, 2678, 2679 MC1_R UniProtKB: V9QBY6 2680 2681 2682, 2683, 2684, 2685, 2686 MC1_R UniProtKB: V9QC17 2687 2688 2689, 2690, 2691, 2692, 2693 MC1_R UniProtKB: V9QC66 2694 2695 2696, 2697, 2698, 2699, 2700 MC1_R UniProtKB: V9QCQ4 2701 2702 2703, 2704, 2705, 2706, 2707 MC1_R UniProtKB: V9QDF4 2708 2709 2710, 2711, 2712, 2713, 2714 MC1_R UniProtKB: V9QDN7 2715 2716 2717, 2718, 2719, 2720, 2721 MC1_R UniProtKB: V9QDQ6 2722 2723 2724, 2725, 2726, 2727, 2728 mesothelin UniProtKB: Q13421 2729 2730 2731, 2732, 2733, 2734, 2735 MITF UniProtKB: O75030-8 2736 2737 2738, 2739, 2740, 2741, 2742 MITF UniProtKB: O75030-9 2743 2744 2745, 2746, 2747, 2748, 2749 MITF UniProtKB: O75030 2750 2751 2752, 2753, 2754, 2755, 2756 MMP1_1 UniProtKB: B3KQS8 2757 2758 2759, 2760, 2761, 2762, 2763 MMP7 UniProtKB: P09237 2764 2765 2766, 2767, 2768, 2769, 2770 MUC-1 Genbank: AAA60019 2771 2772 2773, 2774, 2775, 2776, 2777 MUM-1/m RefSeq: NP_116242 2778 2779 2780, 2781, 2782, 2783, 2784 MUM-2/m UniProtKB: Q9Y5R8 2785 2786 2787, 2788, 2789, 2790, 2791 MYO1A UniProtKB: Q9UBC5 2792 2793 2794, 2795, 2796, 2797, 2798 MYO1B UniProtKB: O43795 2799 2800 2801, 2802, 2803, 2804, 2805 MYO1C UniProtKB: O00159 2806 2807 2808, 2809, 2810, 2811, 2812 MYO1D UniProtKB: O94832 2813 2814 2815, 2816, 2817, 2818, 2819 MYO1E UniProtKB: Q12965 2820 2821 2822, 2823, 2824, 2825, 2826 MYO1F UniProtKB: O00160 2827 2828 2829, 2830, 2831, 2832, 2833 MYO1G UniProtKB: B0I1T2 2834 2835 2836, 2837, 2838, 2839, 2840 MYO1H RefSeq: NP_001094891 2841 2842 2843, 2844, 2845, 2846, 2847 NA17 UniProtKB: Q3V5L5 2848 2849 2850, 2851, 2852, 2853, 2854 NA88-A Pubmed: 10790436 2855 2856 2857, 2858, 2859, 2860, 2861 Neo-PAP UniProtKB: Q9BWT3 2862 2863 2864, 2865, 2866, 2867, 2868 NFYC/m UniProtKB: Q13952 2869 2870 2871, 2872, 2873, 2874, 2875 NGEP UniProtKB: Q6IWH7 2876 2877 2878, 2879, 2880, 2881, 2882 NPM UniProtKB: P06748 2883 2884 2885, 2886, 2887, 2888, 2889 NRCAM UniProtKB: Q92823 2890 2891 2892, 2893, 2894, 2895, 2896 NSE UniProtKB: P09104 2897 2898 2899, 2900, 2901, 2902, 2903 NUF2 UniProtKB: Q9BZD4 2904 2905 2906, 2907, 2908, 2909, 2910 NY-ESO-1 UniProtKB: P78358 2911 2912 2913, 2914, 2915, 2916, 2917 OA1 UniProtKB: P51810 2918 2919 2920, 2921, 2922, 2923, 2924 OGT UniProtKB: O15294 2925 2926 2927, 2928, 2929, 2930, 2931 OS-9 UniProtKB: B4DH11 2932 2933 2934, 2935, 2936, 2937, 2938 OS-9 UniProtKB: B4E321 2939 2940 2941, 2942, 2943, 2944, 2945 OS-9 UniProtKB: B7Z8E7 2946 2947 2948, 2949, 2950, 2951, 2952 OS-9 UniProtKB: Q13438 2953 2954 2955, 2956, 2957, 2958, 2959 osteocalcin UniProtKB: P02818 2960 2961 2962, 2963, 2964, 2965, 2966 osteopontin UniProtKB: A0A024RDE2 2967 2968 2969, 2970, 2971, 2972, 2973 osteopontin UniProtKB: A0A024RDE6 2974 2975 2976, 2977, 2978, 2979, 2980 osteopontin UniProtKB: A0A024RDJ0 2981 2982 2983, 2984, 2985, 2986, 2987 osteopontin UniProtKB: B7Z351 2988 2989 2990, 2991, 2992, 2993, 2994 osteopontin UniProtKB: F2YQ21 2995 2996 2997, 2998, 2999, 3000, 3001 osteopontin UniProtKB: P10451 3002 3003 3004, 3005, 3006, 3007, 3008 p53 UniProtKB: P04637 3009 3010 3011, 3012, 3013, 3014, 3015 PAGE-4 UniProtKB: O60829 3016 3017 3018, 3019, 3020, 3021, 3022 PAI-1 UniProtKB: P05121 3023 3024 3025, 3026, 3027, 3028, 3029 PAI-2 UniProtKB: P05120 3030 3031 3032, 3033, 3034, 3035, 3036 PAP UniProtKB: Q06141 3037 3038 3039, 3040, 3041, 3042, 3043 PAP UniProtKB: Q53S56 3044 3045 3046, 3047, 3048, 3049, 3050 PATE UniProtKB: Q8WXA2 3051 3052 3053, 3054, 3055, 3056, 3057 PAX3 UniProtKB: P23760 3058 3059 3060, 3061, 3062, 3063, 3064 PAX5 UniProtKB: Q02548 3065 3066 3067, 3068, 3069, 3070, 3071 PD1L1 UniProtKB: Q9NZQ7 3072 3073 3074, 3075, 3076, 3077, 3078 PDCD1 UniProtKB: Q15116 3079 3080 3081, 3082, 3083, 3084, 3085 PDEF UniProtKB: O95238 3086 3087 3088, 3089, 3090, 3091, 3092 PECA1 UniProtKB: P16284 3093 3094 3095, 3096, 3097, 3098, 3099 PGCB UniProtKB: Q96GW7 3100 3101 3102, 3103, 3104, 3105, 3106 PGFRB UniProtKB: P09619 3107 3108 3109, 3110, 3111, 3112, 3113 Pim-1_-Kinase UniProtKB: A0A024RD25 3114 3115 3116, 3117, 3118, 3119, 3120 Pin-1 UniProtKB: O15428 3121 3122 3123, 3124, 3125, 3126, 3127 Pin-1 UniProtKB: Q13526 3128 3129 3130, 3131, 3132, 3133, 3134 Pin-1 UniProtKB: Q49AR7 3135 3136 3137, 3138, 3139, 3140, 3141 PLAC1 UniProtKB: Q9HBJ0 3142 3143 3144, 3145, 3146, 3147, 3148 PMEL UniProtKB: P40967 3149 3150 3151, 3152, 3153, 3154, 3155 PML UniProtKB: P29590 3156 3157 3158, 3159, 3160, 3161, 3162 POTEF UniProtKB: A5A3E0 3163 3164 3165, 3166, 3167, 3168, 3169 POTE UniProtKB: Q86YR6 3170 3171 3172, 3173, 3174, 3175, 3176 PRAME UniProtKB: A0A024R1E6 3177 3178 3179, 3180, 3181, 3182, 3183 PRAME UniProtKB: P78395 3184 3185 3186, 3187, 3188, 3189, 3190 PRDX5/m UniProtKB: P30044 3191 3192 3193, 3194, 3195, 3196, 3197 PRM2 UniProtKB: P04554 3198 3199 3200, 3201, 3202, 3203, 3204 prostein UniProtKB: Q96JT2 3205 3206 3207, 3208, 3209, 3210, 3211 proteinase-3 UniProtKB: D6CHE9 3212 3213 3214, 3215, 3216, 3217, 3218 proteinase-3 UniProtKB: P24158 3219 3220 3221, 3222, 3223, 3224, 3225 PSA UniProtKB: P55786 3226 3227 3228, 3229, 3230, 3231, 3232 PSB9 UniProtKB: P28065 3233 3234 3235, 3236, 3237, 3238, 3239 PSCA UniProtKB: D3DWI6 3240 3241 3242, 3243, 3244, 3245, 3246 PSCA UniProtKB: O43653 3247 3248 3249, 3250, 3251, 3252, 3253 PSGR UniProtKB: Q9H255 3254 3255 3256, 3257, 3258, 3259, 3260 PSM UniProtKB: Q04609 3261 3262 3263, 3264, 3265, 3266, 3267 PTPRC RefSeq: NP_002829 3268 3269 3270, 3271, 3272, 3273, 3274 RAB8A UniProtKB: P61006 3275 3276 3277, 3278, 3279, 3280, 3281 RAGE-1 UniProtKB: Q9UQ07 3282 3283 3284, 3285, 3286, 3287, 3288 RARA UniProtKB: P10276 3289 3290 3291, 3292, 3293, 3294, 3295 RASH UniProtKB: P01112 3296 3297 3298, 3299, 3300, 3301, 3302 RASK UniProtKB: P01116 3303 3304 3305, 3306, 3307, 3308, 3309 RASN UniProtKB: P01111 3310 3311 3312, 3313, 3314, 3315, 3316 RGS5 UniProtKB: O15539 3317 3318 3319, 3320, 3321, 3322, 3323 RHAMM/CD168 UniProtKB: O75330 3324 3325 3326, 3327, 3328, 3329, 3330 RHOC UniProtKB: P08134 3331 3332 3333, 3334, 3335, 3336, 3337 RSSA UniProtKB: P08865 3338 3339 3340, 3341, 3342, 3343, 3344 RU1 UniProtKB: Q9UHJ3 3345 3346 3347, 3348, 3349, 3350, 3351 RU2 UniProtKB: Q9UHG0 3352 3353 3354, 3355, 3356, 3357, 3358 RUNX1 UniProtKB: Q01196 3359 3360 3361, 3362, 3363, 3364, 3365 S-100 UniProtKB: V9HW39 3366 3367 3368, 3369, 3370, 3371, 3372 SAGE UniProtKB: Q9NXZ1 3373 3374 3375, 3376, 3377, 3378, 3379 SART-_1 UniProtKB: O43290 3380 3381 3382, 3383, 3384, 3385, 3386 SART-2 UniProtKB: Q9UL01 3387 3388 3389, 3390, 3391, 3392, 3393 SART-3 UniProtKB: Q15020 3394 3395 3396, 3397, 3398, 3399, 3400 SEPR UniProtKB: Q12884 3401 3402 3403, 3404, 3405, 3406, 3407 SIA7F UniProtKB: Q969X2 3408 3409 3410, 3411, 3412, 3413, 3414 SIA8A UniProtKB: Q92185 3415 3416 3417, 3418, 3419, 3420, 3421 SIAT9 UniProtKB: Q9UNP4 3422 3423 3424, 3425, 3426, 3427, 3428 SIRT2/m UniProtKB: A0A024R0G8 3429 3430 3431, 3432, 3433, 3434, 3435 SIRT2/m UniProtKB: Q8IXJ6 3436 3437 3438, 3439, 3440, 3441, 3442 SOX10 UniProtKB: P56693 3443 3444 3445, 3446, 3447, 3448, 3449 SP17 UniProtKB: Q15506 3450 3451 3452, 3453, 3454, 3455, 3456 SPNXA UniProtKB: Q9NS26 3457 3458 3459, 3460, 3461, 3462, 3463 SPXN3 UniProtKB: Q5MJ09 3464 3465 3466, 3467, 3468, 3469, 3470 SSX-1 UniProtKB: Q16384 3471 3472 3473, 3474, 3475, 3476, 3477 SSX-2 UniProtKB: Q16385 3478 3479 3480, 3481, 3482, 3483, 3484 SSX3 UniProtKB: Q99909 3485 3486 3487, 3488, 3489, 3490, 3491 SSX-4 UniProtKB: O60224 3492 3493 3494, 3495, 3496, 3497, 3498 ST1A1 UniProtKB: P50225 3499 3500 3501, 3502, 3503, 3504, 3505 STAG2 UniProtKB: Q8N3U4-2 3506 3507 3508, 3509, 3510, 3511, 3512 STAMP-1 UniProtKB: Q8NFT2 3513 3514 3515, 3516, 3517, 3518, 3519 STEAP-1 UniProtKB: A0A024RA63 3520 3521 3522, 3523, 3524, 3525, 3526 STEAP-1 UniProtKB: Q9UHE8 3527 3528 3529, 3530, 3531, 3532, 3533 Survivin-2B UniProtKB: O15392-2 3534 3535 3536, 3537, 3538, 3539, 3540 survivin UniProtKB: O15392 3541 3542 3543, 3544, 3545, 3546, 3547 SYCP1 UniProtKB: A0A024R0I2 3548 3549 3550, 3551, 3552, 3553, 3554 SYCP1 UniProtKB: B7ZLS9 3555 3556 3557, 3558, 3559, 3560, 3561 SYCP1 UniProtKB: Q15431 3562 3563 3564, 3565, 3566, 3567, 3568 SYCP1 UniProtKB: Q3MHC4 3569 3570 3571, 3572, 3573, 3574, 3575 SYT-SSX-1 UniProtKB: A4PIV7 3576 3577 3578, 3579, 3580, 3581, 3582 SYT-SSX-1 UniProtKB: A4PIV8 3583 3584 3585, 3586, 3587, 3588, 3589 SYT-SSX-2 UniProtKB: A4PIV9 3590 3591 3592, 3593, 3594, 3595, 3596 SYT-SSX-2 UniProtKB: A4PIW0 3597 3598 3599, 3600, 3601, 3602, 3603 TARP UniProtKB: Q0VGM3 3604 3605 3606, 3607, 3608, 3609, 3610 TCRg UniProtKB: A2JGV3 3611 3612 3613, 3614, 3615, 3616, 3617 TF2AA UniProtKB: P52655 3618 3619 3620, 3621, 3622, 3623, 3624 TGFR2 UniProtKB: P37173 3625 3626 3627, 3628, 3629, 3630, 3631 TGM-4 UniProtKB: B2R7D1 3632 3633 3634, 3635, 3636, 3637, 3638 TIE2 UniProtKB: Q02763 3639 3640 3641, 3642, 3643, 3644, 3645 TKTL1 UniProtKB: P51854 3646 3647 3648, 3649, 3650, 3651, 3652 TPI/m UniProtKB: P60174 3653 3654 3655, 3656, 3657, 3658, 3659 TRGV11 UniProtKB: Q99601 3660 3661 3662, 3663, 3664, 3665, 3666 TRGV9 UniProtKB: A4D1X2 3667 3668 3669, 3670, 3671, 3672, 3673 TRGV9 UniProtKB: Q99603 3674 3675 3676, 3677, 3678, 3679, 3680 TRGV9 UniProtKB: Q99604 3681 3682 3683, 3684, 3685, 3686, 3687 TRPC1 UniProtKB: P48995 3688 3689 3690, 3691, 3692, 3693, 3694 TRP-p8 UniProtKB: Q7Z2W7 3695 3696 3697, 3698, 3699, 3700, 3701 TSG10 UniProtKB: Q9BZW7 3702 3703 3704, 3705, 3706, 3707, 3708 TSPY1 UniProtKB: Q01534 3709 3710 3711, 3712, 3713, 3714, 3715 TVC_(TRGV3) Genbank: M13231.1 3716 3717 3718, 3719, 3720, 3721, 3722 TX101 UniProtKB: Q9BY14-2 3723 3724 3725, 3726, 3727, 3728, 3729 tyrosinase UniProtKB: A0A024DBG7 3730 3731 3732, 3733, 3734, 3735, 3736 tyrosinase UniProtKB: L8B082 3737 3738 3739, 3740, 3741, 3742, 3743 tyrosinase UniProtKB: L8B086 3744 3745 3746, 3747, 3748, 3749, 3750 tyrosinase UniProtKB: L8B0B9 3751 3752 3753, 3754, 3755, 3756, 3757 tyrosinase UniProtKB: O75767 3758 3759 3760, 3761, 3762, 3763, 3764 tyrosinase UniProtKB: P14679 3765 3766 3767, 3768, 3769, 3770, 3771 tyrosinase UniProtKB: U3M8N0 3772 3773 3774, 3775, 3776, 3777, 3778 tyrosinase UniProtKB: U3M9D5 3779 3780 3781, 3782, 3783, 3784, 3785 tyrosinase UniProtKB: U3M9J2 3786 3787 3788, 3789, 3790, 3791, 3792 TYRP1 UniProtKB: P17643 3793 3794 3795, 3796, 3797, 3798, 3799 TYRP2 UniProtKB: P40126 3800 3801 3802, 3803, 3804, 3805, 3806 UPA UniProtKB: Q96NZ9 3807 3808 3809, 3810, 3811, 3812, 3813 VEGFR1 UniProtKB: B5A924 3814 3815 3816, 3817, 3818, 3819, 3820 WT1 UniProtKB: A0A0H5AUY0 3821 3822 3823, 3824, 3825, 3826, 3827 WT1 UniProtKB: P19544 3828 3829 3830, 3831, 3832, 3833, 3834 WT1 UniProtKB: Q06250 3835 3836 3837, 3838, 3839, 3840, 3841 XAGE1 UniProtKB: Q9HD64 3842 3843 3844, 3845, 3846, 3847, 3848 IL-10 UniProtKB: P22301 4169 4170 4171, 4172, 4173, 4174, 4175, 4176 IL-5 UniProtKB: P05113 4585 4586 4587, 4588, 4589, 4590, 4591, 4592 M-CSF UniProtKB: P09603 4705 4706 4707, 4708, 4709, 4710, 4711, 4712 TGFbeta1 UniProtKB: P01137 4785 4786 4787, 4788, 4789, 4790, 4791, 4792 Caspase_8 UniProtKB: Q14790 7113 7114 7115, 7116, 7117, 7118, 7119, 7120 SERPINB5 UniProtKB: P36952 7465 7466 7467, 7468, 7469, 7470, 7471, 7472 calreticulin UniProtKB: B4DHR1 7569 7570 7571, 7572, 7573, 7574, 7575, 7576 calreticulin UniProtKB: B4E2Y9 7577 7578 7579, 7580, 7581, 7582, 7583, 7584 calreticulin UniProtKB: P27797 7585 7586 7587, 7588, 7589, 7590, 7591, 7592 calreticulin UniProtKB: Q96L12 7593 7594 7595, 7596, 7597, 7598, 7599, 7600 N-myc UniProtKB: P04198 9987 9988 9989, 9990, 9991, 9992, 9993, 9994

According to the present invention, in a more preferred embodiment, the inventive composition comprises at least one RNA, preferably an mRNA comprising at least one coding region encoding at least one tumor antigen or a fragment or variant thereof, wherein the at least one coding region comprises an RNA sequence being identical or at least 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the RNA sequences according to the SEQ ID Nos as disclosed in Table 9.

Furthermore tumor antigens also may encompass idiotypic antigens associated with a cancer or tumor disease, particularly lymphoma or a lymphoma associated disease, wherein said idiotypic antigen is an immunoglobulin idiotype of a lymphoid blood cell or a T cell receptor idiotype of a lymphoid blood cell.

In a particularly preferred embodiment the inventive RNA composition comprises at least one RNA, wherein the at least one RNA encodes the following antigens:

-   -   STEAP (Six Transmembrane Epithelial Antigen of the Prostate);     -   PSA (Prostate-Specific Antigen),     -   PSMA (Prostate-Specific Membrane Antigen),     -   PSCA (Prostate Stem Cell Antigen);     -   PAP (Prostatic Acid Phosphatase), and     -   MUC1 (Mucin 1).

In another particularly preferred embodiment the inventive RNA composition comprises at least one RNA, wherein the at least one RNA encodes the following antigens:

-   -   5T4 (Trophoblast glycoprotein, TPBG);     -   Survivin (Baculoviral IAP repeat-containing protein 5; BIRC5),     -   NY-ESO-1 (New York esophageal squamous cell carcinoma 1;         CTAG1B),     -   MAGE-C1 (Melanoma antigen family C1);     -   MAGE-C2 (Melanoma antigen family C2), and     -   MUC1 (Mucin 1).

9. β-Catenin Inhibitors

In a further preferred embodiment of the inventive RNA containing composition the RNA, preferably mRNA codes for at least one β-catenin inhibitor or a fragment or variant thereof. Preferably the RNA encoding the at least one β-catenin inhibitor encodes an inhibitory protein or dominant negative mutant protein of the β-catenin pathway. Particular preferred β-catenin inhibitors according to the present invention comprise TAT-NLS-BLBD-6, axin-1, TCF-4, GSK-3b, DKK-1, Dvl-1 derivatives or fragments thereof.

As reviewed by Thakur and Mishra (Thakur R, Mishra D P. Pharmacological modulation of beta-catenin and its applications in cancer therapy. J Cell Mol Med. 2013 April; 17(4):449-56. doi: 10.1111/jcmm.12033) beta-catenin (β-catenin) is a multifunctional protein which plays an important role in physiological homeostasis. It acts both as a transcriptional regulator and an adaptor protein for intracellular adhesion. β-catenin is necessary for the establishment and maintenance of epithelial layers and provides a linkage between intracellular junctions and cytoskeletal proteins. β-catenin is regulated by Wnt signaling. In the absence of Wnt downstream signal β-catenin is phosphorylated which leads to its ubiquitination and eventually protein degradation. Various literature reports have linked β-catenin to the malignant transformation of normal cells. For example, Wnt signaling and β-catenin nuclear localization was associated with differentiation of hepatocytes into a tumoral phenotype. Similarly, in lung epithelial and pancreatic cells, activation of β-catenin was sufficient for induction of oncogenic transformation. In addition to being a driving force of malignant transformation, abnormal β-catenin expression and localization has been associated with increased metastatic potential. Recently, it has been shown that β-catenin signaling prevents T cell infiltration and anti-tumor immunity strongly limiting the potential effects of immunotherapies. Since β-catenin plays an important and detrimental role in tumorigenesis, it has been proposed as a putative drug target.

10. STING-Pathway Activators

In a further preferred embodiment of the inventive RNA containing composition the RNA, preferably mRNA codes for at least one activator of the STING (stimulator of interferon genes) pathway or a fragment or variant thereof. Preferably, the RNA encoding the at least one activator (stimulator) of the STING pathway encodes an activating protein or a constitutively active protein of the STING pathway, preferably DDX41, STING, cGAS, IRF3, TBK1 or STAT6 or a fragment or variant thereof.

As reviewed by Woo et al. (Woo S R, Corrales L, Gajewski T F. The STING pathway and the T cell-inflamed tumor microenvironment. Trends Immunol. 2015 Mar. 7. pii: S1471-4906(15)00019-8. doi: 10.1016/j.it.2015.02.003) and Dubensky et al. (Dubensky T W Jr, Kanne D B, Leong M L. Rationale, progress and development of vaccines utilizing STING-activating cyclic dinucleotide adjuvants. Ther Adv Vaccines. 2013 November; 1(4):131-43. doi: 10.1177/2051013613501988) the so-called STING pathway (STING—stimulator of interferon genes) is responsible for sensing of cytoplasmic DNA and induction of proinflammatory mediators. After binding of DNA in cytoplasm, STING activates signaling via TANK-binding kinase 1 (TBK-1)/IRF-3 axis which results in production of IFN-β. This pathway was shown to play an important role in sensing of DNA viruses as well as some autoimmune disorders. Recent data have identified STING pathway as absolutely necessary to induce spontaneous T cell priming against tumor antigens in vivo. Tumor DNA was detected within tumor-infiltrating DCs, which led to IFN-β production and T cell activation. Thus, intratumoral application of small molecules STING pathway agonists has demonstrated their efficacy in tumor-bearing animals. Agonists of the STING pathway has been also evaluated as vaccine adjuvants showing potency to induce cellular and humoral immunity in vaccinated hosts.

11. Checkpoint Modulators

In a further preferred embodiment of the inventive RNA containing composition the RNA, preferably mRNA comprises at least one coding region that codes for at least one checkpoint modulator or a fragment or variant thereof.

Negative regulatory T cell surface molecules were discovered which are upregulated in activated T cells to dampen their activity, resulting in less effective killing of tumor cells. These inhibitory molecules were termed negative co-stimulatory molecules due to their homology to the T cell co-stimulatory molecule CD28. These proteins, also referred to as immune checkpoint proteins, function in multiple pathways including the attenuation of early activation signals, competition for positive co-stimulation and direct inhibition of antigen presenting cells (Bour-Jordan et al., 2011. Immunol Rev. 241(1):180-205).

In preferred embodiments of the present invention the checkpoint modulator is a modulator of B7-1/CD80, B7-2/CD86, B7-H1/PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7/HHLA2, BTLA, CD28, CD28H/IGPR-1, CTLA-4, ICOS, PD-1, PD-L2/B7-DC, PDCD6, VISTA/B7-H5/PD-1H, BTN1A1/Butyrophilin, BTN2A1, BTN2A2/Butyrophilin 2A2, BTN3A1/2, BTN3A2, BTN3A3, BTNL2/Butyrophilin-like 2, BTNL3, BTNL4, BTNL6, BTNL8, BTNL9, BTNL10, CD277/BTN3A1, LAIR1, LAIR2, CD96, CD155/PVR, CRTAM, DNAM-1/CD226, Nectin-2/CD112, Nectin-3, TIGIT, LILRA3/CD85e, LILRA4/CD85g/ILT7, LILRB1/CD85j/ILT2, LILRB2/CD85d/ILT4, LILRB3/CD85a/ILT5, LILRB4/CD85k/ILT3, 4-1BB/TNFRSF9/CD137, 4-1BB Ligand/TNFSF9, BAFF/BLyS/TNFSF13B, BAFF R/TNFRSF13C, CD27/TNFRSF7, CD27 Ligand/TNFSF7, CD30/TNFRSF8, CD30 Ligand/TNFSF8, CD40/TNFRSF5, CD40 Ligand/TNFSF5, DR3/TNFRSF25, GITR/TNFRSF18, GITR Ligand/TNFSF18, HVEM/TNFRSF14, LIGHT/TNFSF14, Lymphotoxin-alpha/TNF-beta, OX40/TNFRSF4, OX40 Ligand/TNFSF4, RELT/TNFRSF19L, TACI/TNFRSF13B, TL1A/TNFSF15, TNF-alpha, TNF RII/TNFRSF1B, 2B4/CD244/SLAMF4, BLAME/SLAMF8, CD2, CD2F-10/SLAMF9, CD48/SLAMF2, CD58/LFA-3, CD84/SLAMF5, CD229/SLAMF3, CRACC/SLAMF7, NTB-A/SLAMF6, SLAM/CD150, TIM-1/KIM-1/HAVCR, TIM-3, TIM-4, CD7, CD96, CD160, CD200, CD300a/LMIR1, CRTAM, DAP12, Dectin-1/CLEC7A, DPPIV/CD26, EphB6, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM-1, LAG-3, TIM-1/KIM-1/HAVCR, TIM-4, TSLP R, or any combinations thereof.

In the context of the present invention a checkpoint modulator is defined herein as a molecule preferably a protein e.g. an antibody, a dominant negative receptor, a decoy receptor, or a ligand or a fragment or variant thereof, which modulates the function of an immune checkpoint protein, e.g. it inhibits or reduces the activity of checkpoint inhibitors (or inhibitory checkpoint molecules) or it stimulates the activity of checkpoint stimulators (or stimulatory checkpoint molecules). Therefore checkpoint modulators as defined herein, influence the activity of checkpoint molecules.

In this context inhibitory checkpoint molecules are defined as checkpoint inhibitors and can be used synonymously. In addition stimulatory checkpoint molecules are defined as checkpoint stimulators and can be used synonymously.

Preferable inhibitory checkpoint molecules that may be inhibited by a checkpoint modulator in the context of the invention are PD-1, PD-L1, CTLA-4, PD-L2, LAG3, TIM3/HAVCR2, 2B4, A2aR, B7H3, B7H4, BTLA, CD30, CD160, GAL9, HVEM, IDO1, IDO2, KIR, LAIR1 and VISTA.

Preferable stimulatory checkpoint molecules that may be stimulated by a checkpoint modulator in the context of the invention are CD2, CD27, CD28, CD40, CD137, CD226, CD276, GITR, ICOS, OX-40 and CD70.

Preferably, the checkpoint modulator is selected from agonistic antibodies, antagonistic antibodies, ligands, dominant negative receptors, and decoy receptors or combinations thereof.

Methods for generating and using mRNA-encoded antibodies are known in the art (e.g. WO2008/083949).

Preferably, the agonistic antibody is chosen from the following list: anti-4-1BB, anti-OX40, anti-GITR, anti-CD28, anti-CD27, anti-CD-40anti-ICOS, anti-TNFRSF25, and anti-LIGHT.

OX40 is a member of the TNFR-superfamily of receptors, and is expressed on the surface of antigen-activated mammalian CD4+ and CD8+ T lymphocytes. OX40 ligand (OX40L, also known as gp34, ACT-4-L, and CD252) is a protein that specifically interacts with the OX40 receptor. The term OX40L includes the entire OX40 ligand, soluble OX40 ligand, and fusion proteins comprising a functionally active portion of OX40 ligand covalently linked to a second moiety, e.g., a protein domain. Also included within the definition of OX40L are variants which vary in amino acid sequence from naturally occurring OX4L but which retain the ability to specifically bind to the OX40 receptor. Further included within the definition of OX40L are variants which enhance the biological activity of OX40. An OX40 agonist is a molecule which induces or enhances the biological activity of OX40, e.g. signal transduction mediated by OX40. An OX40 agonist is preferably defined herein as a binding molecule capable of specific binding to OX40. Therefore, the OX40 agonist may be any agonist binding to OX40 and capable of stimulating OX40 signaling. In this context, the OX40 agonist may be an agonistic antibody binding to OX40.

OX40 agonists and anti-OX40 monoclonal antibodies are described in WO1995/021251, WO1995/012673 and WO1995/21915. Particularly preferred is the anti-OX40 antibody 9B12, a murine anti-OX40 monoclonal antibody directed against the extracellular domain of human OX40 (Weinberg et al., 2006. J. Immunother. 29(6):575-585).

Preferably, the antagonistic antibody is chosen from the list of anti-CTLA4, anti-PD1, anti-PD-L1, anti-Vista, anti-Tim-3, anti-LAG-3, and anti-BTLA.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is mainly expressed within the intracellular compartment of T cells. After a potent or long-lasting stimulus to a naive T cell via the T cell receptor (TCR), CTLA-4 is transported to the cell surface and concentrated at the immunological synapse. CTLA-4 then competes with CD28 for CD80/CD86 and down-modulates TCR signaling via effects on Akt signaling. Thus CTLA-4 functions physiologically as a signal dampener (Weber, J. 2010. Semin. Oncol. 37(5):430-9).

Particularly preferred are the anti-CTLA-4 antibodies ipilimumab (Yervoy®), tremelimumab, and AGEN-1884.

Members of the PD-1 pathway are all proteins which are associated with PD-1 signaling. On the one hand these might be proteins which induce PD-1 signaling upstream of PD-1 as e.g. the ligands of PD-1 PD-L1 and PD-L2 and the signal transduction receptor PD-1. On the other hand these might be signal transduction proteins downstream of PD-1 receptor. Particularly preferred as members of the PD-1 pathway in the context of the present invention are PD-1, PD-L1 and PD-L2.

In the context of the present invention, a PD-1 pathway antagonist is preferably defined herein as a compound capable to impair the PD-1 pathway signaling, preferably signaling mediated by the PD-1 receptor. Therefore, the PD-1 pathway antagonist may be any antagonist directed against any member of the PD-1 pathway capable of antagonizing PD-1 pathway signaling. In this context, the antagonist may be an antagonistic antibody as defined herein, targeting any member of the PD-1 pathway, preferably directed against PD-1 receptor, PD-L1 or PD-L2. This antagonistic antibody may also be encoded by a nucleic acid. Also, the PD-1 pathway antagonist may be a fragment of the PD-1 receptor blocking the activity of PD1 ligands. B7-1 or fragments thereof may act as PD1-antagonizing ligands as well. Additionally, a PD-1 pathway antagonist may be a protein comprising (or a nucleic acid coding for) an amino acid sequence capable of binding to PD-1 but preventing PD-1 signaling, e.g. by inhibiting PD-1 and B7-H1 or B7-DL interaction (WO2014127917).

Particularly preferred are the anti-PD1 antibodies Nivolumab (MDX-1106/BMS-936558/ONO-4538), (Brahmer et al., 2010. J Clin Oncol. 28(19):3167-75; PMID: 20516446); Pidilizumab (CT-011), (Berger et al., 2008. Clin Cancer Res. 14(10):3044-51; PMID: 18483370); Pembrolizumab (MK-3475, SCH 900475); AMP-224, and MED10680 (AMP-514)

Particularly preferred are the anti-PD-L1 antibodies MDX-1105/BMS-936559 (Brahmer et al. 2012. N Engl J Med. 366(26):2455-65; PMID: 22658128); atezolizumab (MPDL3280A/RG7446); durvalumab (MED14736); and avelumab (MSB0010718).

According to the present invention the at least one RNA of the inventive RNA containing composition encodes at least one antibody or fragments or variants thereof of Table 10. It is particularly preferred that the RNA containing composition comprises at least one RNA encoding the heavy chain of a particular antibody or fragments or variants thereof and at least one further RNA encoding the light chain of the same particular antibody or fragments or variants thereof.

TABLE 10 Antibodies directed against checkpoint molecules Name Target Urelumab 4-1BB/CD137 PF-05082566 4-1BB/CD137 8H9 B7-H3 Enoblituzumab B7-H3 Ipilimumab CD152/CTLA-4 Ticilimumab (=tremelimumab) CD152/CTLA-4 Tremelimumab CD152/CTLA-4 Varlilumab CD27 Teneliximab CD40 Vorsetuzumab mafodotin CD70 Lirilumab KIR2D GSK-3174998 OX40 MEDI-6469 OX40 MEDI-6383 OX40 MEDI-0562 OX40 PF-04518600 OX40 RG-7888 OX40 PF-06801591 PD-1 BGBA-317 PD-1 MEDI-0680 PD-1 MK-3475 PD-1 Nivolumab PD-1 PDR-001 PD-1 Pembrolizumab PD-1 Pidilizumab PD-1 REGN-2810 PD-1 SHR-1210 PD-1 TSR-042 PD-1 MDX-1106 PD-1 Merck 3745 PD-1 CT-011 PD-1 MEDI-0680 PD-1 PDR001 PD-1 REGN2810 PD-1 BGB-108 PD-1 BGB-A317 PD-1 AMP-224 PD-1 Atezolizumab PD-L1 (CD274) Avelumab PD-L1 (CD274) BMS-936559 PD-L1 (CD274) Durvalumab PD-L1 (CD274) MEDI-4736 PD-L1 (CD274) MPDL33280A PD-L1 (CD274) YW243.55.S70 PD-L1 (CD274) MDX-1105 PD-L1 (CD274) MSB0010718C PD-L1 (CD274)

In a further preferred embodiment the checkpoint modulator is a decoy receptor (e.g. a soluble receptor). Preferably, the decoy receptor is a soluble PD1 receptor. In a particularly preferred embodiment the at least one RNA of the inventive RNA containing composition comprises an RNA sequence being identical or at least 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the RNA sequence according to SEQ ID NO: 389 encoding a soluble PD-1 receptor.

In a further preferred embodiment of the inventive RNA containing composition the RNA, preferably an mRNA codes for at least one ligand which functions as a checkpoint modulator. Preferably, the ligand is CD40 Ligand (CD40L). In a further preferred embodiment of the inventive RNA containing composition the RNA, preferably an mRNA codes for at least one ligand which functions as a checkpoint modulator. Preferably, the ligand is CD40 Ligand (CD40L). Most preferably the at least one RNA of the inventive RNA containing composition comprises an RNA sequence being identical or at least 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the RNA sequence according to SEQ ID NO: 10073 encoding CD40L.

12. Innate Immune Activators:

In this context innate immune activators may be selected from mammalian, in particular human adjuvant proteins, which typically comprise any human protein or peptide, which is capable of eliciting an innate immune response (in a mammal), e.g. as a reaction of the binding of an exogenous TLR ligand to a TLR. More preferably, human adjuvant proteins are selected from the group consisting of proteins which are components and ligands of the signalling networks of the pattern recognition receptors including TLR, NLR and RLH, including TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11; NOD1, NOD2, NOD3, NOD4, NOD5, NALP1, NALP2, NALP3, NALP4, NALP5, NALP6, NALP6, NALP7, NALP7, NALP8, NALP9, NALP10, NALP11, NALP12, NALP13, NALP14, I IPAF, NAIP, CIITA, RIG-I, MDA5 and LGP2, the signal transducers of TLR signaling including adaptor proteins including e.g. Trif and Cardif; components of the Small-GTPases signalling (RhoA, Ras, Rac1, Cdc42, Rab etc.), components of the PIP signalling (PI3K, Src-Kinases, etc.), components of the MyD88-dependent signalling (MyD88, IRAK1, IRAK2, IRAK4, TIRAP, TRAF6 etc.), components of the MyD88-independent signalling (TICAM1, TICAM2, TRAF6, TBK1, IRF3, TAK1, IRAK1 etc.); the activated kinases including e.g. Akt, MEKK1, MKK1, MKK3, MKK4, MKK6, MKK7, ERK1, ERK2, GSK3, PKC kinases, PKD kinases, GSK3 kinases, JNK, p38MAPK, TAK1, IKK, and TAK1; the activated transcription factors including e.g. NF-κB, c-Fos, c-Jun, c-Myc, CREB, AP-1, Elk-1, ATF2, IRF-3, IRF-7.

Mammalian, in particular human adjuvant proteins may furthermore be selected from the group consisting of heat shock proteins, such as HSP10, HSP60, HSP65, HSP70, HSP75 and HSP90, gp96, Fibrinogen, TypIII repeat extra domain A of fibronectin; or components of the complement system including C1q, MBL, C1r, C1s, C2b, Bb, D, MASP-1, MASP-2, C4b, C3b, C5a, C3a, C4a, C5b, C6, C7, C8, C9, CR1, CR2, CR3, CR4, C1qR, C1INH, C4bp, MCP, DAF, H, I, P and CD59, or induced target genes including e.g. Beta-Defensin, cell surface proteins; or human adjuvant proteins including trif, flt-3 ligand, Gp96 or fibronectin, etc., or any species homolog of any of the above human adjuvant proteins. Furthermore HGMB1 may be used as adjuvant protein.

Mammalian, in particular human adjuvant proteins may furthermore comprise cytokines which induce or enhance an innate immune response, including IL-1 alpha, IL1 beta, IL-2, IL-6, IL-7, IL-8, IL-9, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21, IL-23, TNFalpha, IFNalpha, IFNbeta, IFNgamma, GM-CSF, G-CSF, M-CSF; chemokines including IL-8, IP-10, MCP-1, MIP-1alpha, RANTES, Eotaxin, CCL21; cytokines which are released from macrophages, including IL-1, IL-6, IL-8, IL-12 and TNF-alpha; as well as IL-1R1 and IL-1 alpha.

Therefore in this context it particularly preferred that the at least one RNA encodes at least one innate immune activator, preferably an adjuvant protein, more preferably a human adjuvant protein, or a fragment or variant thereof.

In this context it is particularly preferred that I constitutive active variant of an adjuvant protein is encoded by the at least one RNA, preferably a constitutive active variant of RIG-1 (ΔRIGI).

In another preferred embodiment the at least one RNA encodes HGMB1 as an innate immune activator, or a fragment or variant thereof.

According to preferred embodiments in the context of the present invention innate immune activators may be selected from any innate immune activator selected from the group consisting of CD55; Akt; ATF2; C1QBP; C1QC; Cardif; CCL11; CCL2; CCL21; CCL3; CCL5; CD59, Beta-Defensin; Cdc42; CFAD; CFAH; CFAI; CH60; CIITA; c-Jun; c-myc; CO8A; COBB; CO8G; complement_system_component_C1INH; complement_system_component_C1qR; complement_system_component_C1s; complement_system_component_C4bp; complement_system_component_C6; complement_system_component_C7; complement_system_component_C8; complement_system_component_C9; complement_system_component_CR2; complement_system_component_CR3; complement_system_component_MASP-1; complement_system_component_MASP-2; complement_system_component_MBL; complement_system_component_MCP; CREB3; CREB3L1; CREB3L3; CREB3L4; CREB5; CRTC2; CXCL10; CXCL8; DJB11; DJB13; DJB14; DJC10; DJC12; DJC14; DJC15; DJC16; DJC17; DJC18; DJC22; DJC24; DJC25; DJC27; DJC28; DJC30; DNAJB12; DNAJC11; DNAJC21; DNJA1; DNJA2; DNJA3; DNJA4; DNJB1; DNJB2; DNJB3; DNJB4; DNJB5; DNJB6; DNJB7; DNJB8; DNJB9; DNJC1; DNJC2; DNJC3; DNJC4; DNJC5; DNJC7; DNJC8; DNJC9; Elk-1; ERK1; ERK2; Fibrinogen; fibronectin; FLT3_Ligand; FOS; G-CSF; GM-CSF; GRP94_(gp96); GSK3A; GSK3B; HS71A; HS71B; HSC70; HSP10; HSP60; HSP70; HSP75; HSP90; HSP90B1; IFNalpha; IFNB; IFNG; IKK; IL-1; IL-1_alpha; IL-1_beta; IL-12; IL-13; IL-15; IL-16; IL-17A; IL-18; IL-1R1; IL-2; IL-21; IL-23; IL-6; IL-7; IL-9; IRAK1; IRAK2; IRAK4; IRF3; IRF-7; JNK; KPCB; KPCD; KPCD1; KPCD3; KPCE; KPCG; KPCI; KPCL; KPCT; KPCZ; I_IPAF; LGP2; M-CSF; MDA5; MK11; MK12; MK13; MK14; MKK1; MKK3; MKK4; MKK6; MKK7; MSTP104; MyD88; NALP10; NALP11; NALP12; NALP13; NALP2; NALP3; NALP4; NALP5; NALP6; NALP7; NALP8; NALP9; NF-kappaB; NLRP14; NOD1; NOD2; NOD3; PI3K; PKD2; PKN1; PKN2; PKN3; PRKCA; PRKD2; Rab; Rac1; RASH; RASK; RASN; RhoA; RIG-I; Src-Kinases; Surfactant_protein_A; Surfactant_protein_D; TAK1; TBK1; TICAM1; TICAM2; TIRAP; TLR1; TLR10; TLR2; TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9; TNF; TRAF6, preferably as disclosed in Table 11. Particularly preferred in this context are the RNA sequences encoding a innate immune activator according to Table 11.

TABLE 11 Innate immune activators (human adjuvant proteins) RNA Protein Sequence Sequence wild type Protein SEQ ID SEQ ID Optimized RNA Sequence Gene Name Accession No. NO: NO: SEQ ID NO: CD55 UniProtKB: 909 910 911, 912, 913, 914, 915 B1AP15 CD55 UniProtKB: 916 917 918, 919, 920, 921, 922 D3DT85 CD55 UniProtKB: 923 924 925, 926, 927, 928, 929 D3DT86 CD55 UniProtKB: 930 931 932, 933, 934, 935, 936 P08174 fibronectin UniProtKB: 1567 1568 1569, 1570, 1571, 1572, 1573 A0A024R516 fibronectin UniProtKB: 1574 1575 1576, 1577, 1578, 1579, 1580 A0A024111301 fibronectin UniProtKB: 1581 1582 1583, 1584, 1585, 1586, 1587 A0A024RDT9 fibronectin UniProtKB: 1588 1589 1590, 1591, 1592, 1593, 1594 A0A024RDV5 fibronectin UniProtKB: 1595 1596 1597, 1598, 1599, 1600, 1601 A6NH44 fibronectin UniProtKB: 1602 1603 1604, 1605, 1606, 1607, 1608 A8K6A5 fibronectin UniProtKB: 1609 1610 1611, 1612, 1613, 1614, 1615 B2R627 fibronectin UniProtKB: 1616 1617 1618, 1619, 1620, 1621, 1622 B3KXM5 fibronectin UniProtKB: 1623 1624 1625, 1626, 1627, 1628, 1629 B4DIC5 fibronectin UniProtKB: 1630 1631 1632, 1633, 1634, 1635, 1636 B4DN21 fibronectin UniProtKB: 1637 1638 1639, 1640, 1641, 1642, 1643 B4DS98 fibronectin UniProtKB: 1644 1645 1646, 1647, 1648, 1649, 1650 B4DTH2 fibronectin UniProtKB: 1651 1652 1653, 1654, 1655, 1656, 1657 B4DTK1 fibronectin UniProtKB: 1658 1659 1660, 1661, 1662, 1663, 1664 B4DU16 fibronectin UniProtKB: 1665 1666 1667, 1668, 1669, 1670, 1671 B7Z3W5 fibronectin UniProtKB: 1672 1673 1674, 1675, 1676, 1677, 1678 B7Z939 fibronectin UniProtKB: 1679 1680 1681, 1682, 1683, 1684, 1685 G5E9X3 fibronectin UniProtKB: 1686 1687 1688, 1689, 1690, 1691, 1692 Q9H382 FOS UniProtKB: 1693 1694 1695, 1696, 1697, 1698, 1699 P01100 HS71A UniProtKB: 1938 1939 1940, 1941, 1942, 1943, 1944 P0DMV8 HS71B UniProtKB: 1945 1946 1947, 1948, 1949, 1950, 1951 P0DMV9 RASH UniProtKB: 3296 3297 3298, 3299, 3300, 3301, 3302 P01112 RASK UniProtKB: 3303 3304 3305, 3306, 3307, 3308, 3309 P01116 RASN UniProtKB: 3310 3311 3312, 3313, 3314, 3315, 3316 P01111 FLT3 Jigand Genbank: 3913 3914 3915, 3916, 3917, 3918, 3919, AAA90950.1 3920 FLT3 Jigand UniProtKB: 3921 3922 3923, 3924, 3925, 3926, 3927, P49771 3928 G-CSF UniProtKB: 3929 3930 3931, 3932, 3933, 3934, 3935, P09919 3936 GM-CSF UniProtKB: 3945 3946 3947, 3948, 3949, 3950, 3951, P04141 3952 IFNalpha UniProtKB: 3953 3954 3955, 3956, 3957, 3958, 3959, G9JKF1 3960 IFNalpha UniProtKB: 3961 3962 3963, 3964, 3965, 3966, 3967, P01562 3968 IFNalpha UniProtKB: 3969 3970 3971, 3972, 3973, 3974, 3975, P01563 3976 IFNalpha UniProtKB: 3977 3978 3979, 3980, 3981, 3982, 3983, P01566 3984 IFNalpha UniProtKB: 3985 3986 3987, 3988, 3989, 3990, 3991, P01567 3992 IFNalpha UniProtKB: 3993 3994 3995, 3996, 3997, 3998, 3999, P01568 4000 IFNalpha UniProtKB: 4001 4002 4003, 4004, 4005, 4006, 4007, P01569 4008 IFNalpha UniProtKB: 4009 4010 4011, 4012, 4013, 4014, 4015, P01570 4016 IFNalpha UniProtKB: 4017 4018 4019, 4020, 4021, 4022, 4023, P01571 4024 IFNalpha UniProtKB: 4025 4026 4027, 4028, 4029, 4030, 4031, P05013 4032 IFNalpha UniProtKB: 4033 4034 4035, 4036, 4037, 4038, 4039, P05014 4040 IFNalpha UniProtKB: 4041 4042 4043, 4044, 4045, 4046, 4047, P05015 4048 IFNalpha UniProtKB: 4049 4050 4051, 4052, 4053, 4054, 4055, P32881 4056 IFNalpha UniProtKB: 4057 4058 4059, 4060, 4061, 4062, 4063, Q14618 4064 IFNalpha UniProtKB: 4065 4066 4067, 4068, 4069, 4070, 4071, Q86UP4 4072 IFNB UniProtKB: 4073 4074 4075, 4076, 4077, 4078, 4079, P01574 4080 IFNB UniProtKB: 4081 4082 4083, 4084, 4085, 4086, 4087, Q15943 4088 IFNG UniProtKB: 4089 4090 4091, 4092, 4093, 4094, 4095, P01579 4096 IFNG UniProtKB: 4097 4098 4099, 4100, 4101, 4102, 4103, Q14609 4104 IFNG UniProtKB: 4105 4106 4107, 4108, 4109, 4110, 4111, Q14610 4112 IFNG UniProtKB: 4113 4114 4115, 4116, 4117, 4118, 4119, Q14611 4120 IFNG UniProtKB: 4121 4122 4123, 4124, 4125, 4126, 4127, Q14612 4128 IFNG UniProtKB: 4129 4130 4131, 4132, 4133, 4134, 4135, Q14613 4136 IFNG UniProtKB: 4137 4138 4139, 4140, 4141, 4142, 4143, Q14614 4144 IFNG UniProtKB: 4145 4146 4147, 4148, 4149, 4150, 4151, Q14615 4152 IFNG UniProtKB: 4153 4154 4155, 4156, 4157, 4158, 4159, Q8NHY9 4160 IL-12 UniProtKB: 4193 4194 4195, 4196, 4197, 4198, 4199, P29460 4200 IL-13 UniProtKB: 4201 4202 4203, 4204, 4205, 4206, 4207, P35225 4208 IL-15 UniProtKB: 4217 4218 4219, 4220, 4221, 4222, 4223, P40933 4224 IL-16 UniProtKB: 4225 4226 4227, 4228, 4229, 4230, 4231, Q14005 4232 IL-17A UniProtKB: 4233 4234 4235, 4236, 4237, 4238, 4239, Q16552 4240 IL-18 UniProtKB: 4281 4282 4283, 4284, 4285, 4286, 4287, A0A024113E0 4288 IL-18 UniProtKB: 4289 4290 4291, 4292, 4293, 4294, 4295, B0YJ28 4296 IL-18 UniProtKB: 4297 4298 4299, 4300, 4301, 4302, 4303, Q14116 4304 IL-1_alpha UniProtKB: 4313 4314 4315, 4316, 4317, 4318, 4319, P01583 4320 IL-1_beta UniProtKB: 4321 4322 4323, 4324, 4325, 4326, 4327, P01584 4328 IL-21 RefSeq: 4361 4362 4363, 4364, 4365, 4366, 4367, NP_001193935.1 4368 IL-21 RefSeq: 4369 4370 4371, 4372, 4373, 4374, 4375, NP_068575.1 4376 IL-23 UniProtKB: 4385 4386 4387, 4388, 4389, 4390, 4391, Q9NPF7 4392 IL-2 UniProtKB: 4473 4474 4475, 4476, 4477, 4478, 4479, P60568 4480 IL-2 UniProtKB: 4481 4482 4483, 4484, 4485, 4486, 4487, Q0GK43 4488 IL-2 UniProtKB: 4489 4490 4491, 4492, 4493, 4494, 4495, Q13169 4496 IL-2 UniProtKB: 4497 4498 4499, 4500, 4501, 4502, 4503, Q6NZ91 4504 IL-2 UniProtKB: 4505 4506 4507, 4508, 4509, 4510, 4511, Q6NZ93 4512 IL-6 UniProtKB: 4593 4594 4595, 4596, 4597, 4598, 4599, P05231 4600 IL-7 UniProtKB: 4601 4602 4603, 4604, 4605, 4606, 4607, A8K673 4608 IL-7 UniProtKB: 4609 4610 4611, 4612, 4613, 4614, 4615, P13232 4616 IL-9 UniProtKB: 4617 4618 4619, 4620, 4621, 4622, 4623, P15248 4624 M-CSF UniProtKB: 4705 4706 4707, 4708, 4709, 4710, 4711, P09603 4712 CCL11 UniProtKB: 4833 4834 4835, 4836, 4837, 4838, 4839, P51671 4840 CCL11 UniProtKB: 4841 4842 4843, 4844, 4845, 4846, 4847, Q619T4 4848 CCL21 UniProtKB: 4937 4938 4939, 4940, 4941, 4942, 4943, O00585 4944 CCL2 UniProtKB: 5001 5002 5003, 5004, 5005, 5006, 5007, P13500 5008 CCL3 UniProtKB: 5009 5010 5011, 5012, 5013, 5014, 5015, A0N0R1 5016 CCL3 UniProtKB: 5017 5018 5019, 5020, 5021, 5022, 5023, P10147 5024 CCL5 UniProtKB: 5041 5042 5043, 5044, 5045, 5046, 5047, D0EI67 5048 CCL5 UniProtKB: 5049 5050 5051, 5052, 5053, 5054, 5055, P13501 5056 CXCL10 UniProtKB: 5129 5130 5131, 5132, 5133, 5134, 5135, A0A024RDA4 5136 CXCL10 UniProtKB: 5137 5138 5139, 5140, 5141, 5142, 5143, P02778 5144 CXCL8 UniProtKB: 5265 5266 5267, 5268, 5269, 5270, 5271, P10145 5272 TNF UniProtKB: 7369 7370 7371, 7372, 7373, 7374, 7375, P01375 7376 TNF UniProtKB: 7377 7378 7379, 7380, 7381, 7382, 7383, Q5STB3 7384 GRP94_(gp96) UniProtKB: 7617 7618 7619, 7620, 7621, 7622, 7623, P14625 7624 HSC70 UniProtKB: 7625 7626 7627, 7628, 7629, 7630, 7631, P11142 7632 HSP60 UniProtKB: 7657 7658 7659, 7660, 7661, 7662, 7663, A0A024R3X4 7664 HSP60 UniProtKB: 7665 7666 7667, 7668, 7669, 7670, 7671, B3GQS7 7672 HSP60 UniProtKB: 7673 7674 7675, 7676, 7677, 7678, 7679, P10809 7680 HSP60 UniProtKB: 7681 7682 7683, 7684, 7685, 7686, 7687, Q0VDF9 7688 HSP70 UniProtKB: 7689 7690 7691, 7692, 7693, 7694, 7695, P38646 7696 HSP90 UniProtKB: 7697 7698 7699, 7700, 7701, 7702, 7703, P07900 7704 HSP90 UniProtKB: 7705 7706 7707, 7708, 7709, 7710, 7711, P08238 7712 Akt UniProtKB: 7737 7738 7739, 7740, 7741, 7742, 7743 B0LPE5 Akt UniProtKB: 7744 7745 7746, 7747, 7748, 7749, 7750 P31749 Akt UniProtKB: 7751 7752 7753, 7754, 7755, 7756, 7757 P31751 Akt UniProtKB: 7758 7759 7760, 7761, 7762, 7763, 7764 Q9Y243 ATF2 UniProtKB: 7765 7766 7767, 7768, 7769, 7770, 7771 P15336 C1QBP UniProtKB: 7772 7773 7774, 7775, 7776, 7777, 7778 Q07021 C1QC UniProtKB: 7779 7780 7781, 7782, 7783, 7784, 7785 P02747 Cardif UniProtKB: 7786 7787 7788, 7789, 7790, 7791, 7792 Q72434 CD59, Beta-Defensin UniProtKB: 7793 7794 7795, 7796, 7797, 7798, 7799 P13987 CD59, Beta-Defensin UniProtKB: 7800 7801 7802, 7803, 7804, 7805, 7806 Q6FHM9 Cdc42 UniProtKB: 7807 7808 7809, 7810, 7811, 7812, 7813 A0A024RAE4 Cdc42 UniProtKB: 7814 7815 7816, 7817, 7818, 7819, 7820 A0A024RAE6 Cdc42 UniProtKB: 7821 7822 7823, 7824, 7825, 7826, 7827 P60953 CFAD UniProtKB: 7828 7829 7830, 7831, 7832, 7833, 7834 P00746 CFAH UniProtKB: 7835 7836 7837, 7838, 7839, 7840, 7841 P08603 CFAI UniProtKB: 7842 7843 7844, 7845, 7846, 7847, 7848 P05156 CH60 RefSeq: 7849 7850 7851, 7852, 7853, 7854, 7855 NP_002147.2 CIITA UniProtKB: 7856 7857 7858, 7859, 7860, 7861, 7862 Q29704 c-Jun UniProtKB: 7863 7864 7865, 7866, 7867, 7868, 7869 B3KN68 c-Jun UniProtKB: 7870 7871 7872, 7873, 7874, 7875, 7876 B3KNW1 c-Jun UniProtKB: 7877 7878 7879, 7880, 7881, 7882, 7883 B3KXW5 c-Jun UniProtKB: 7884 7885 7886, 7887, 7888, 7889, 7890 B4DED9 c-Jun UniProtKB: 7891 7892 7893, 7894, 7895, 7896, 7897 B4DFU7 c-Jun UniProtKB: 7898 7899 7900, 7901, 7902, 7903, 7904 B4DGE1 c-Jun UniProtKB: 7905 7906 7907, 7908, 7909, 7910, 7911 B4DJ64 c-Jun UniProtKB: 7912 7913 7914, 7915, 7916, 7917, 7918 B4DS36 c-Jun UniProtKB: 7919 7920 7921, 7922, 7923, 7924, 7925 B7Z1L7 c-Jun UniProtKB: 7926 7927 7928, 7929, 7930, 7931, 7932 G1U124 c-Jun UniProtKB: 7933 7934 7935, 7936, 7937, 7938, 7939 G5E966 c-Jun UniProtKB: 7940 7941 7942, 7943, 7944, 7945, 7946 O75843 c-Jun UniProtKB: 7947 7948 7949, 7950, 7951, 7952, 7953 P05412 c-Jun UniProtKB: 7954 7955 7956, 7957, 7958, 7959, 7960 P53677 c-Jun UniProtKB: 7961 7962 7963, 7964, 7965, 7966, 7967 P61966 c-Jun UniProtKB: 7968 7969 7970, 7971, 7972, 7973, 7974 Q63HQ0 c-Jun UniProtKB: 7975 7976 7977, 7978, 7979, 7980, 7981 Q7Z5Q8 c-Jun UniProtKB: 7982 7983 7984, 7985, 7986, 7987, 7988 Q96PC3 c-Jun UniProtKB: 7989 7990 7991, 7992, 7993, 7994, 7995 Q9BXS5 c-Jun UniProtKB: 7996 7997 7998, 7999, 8000, 8001, 8002 Q9Y6Q5 CO8A UniProtKB: 8003 8004 8005, 8006, 8007, 8008, 8009 P07357 COBB UniProtKB: 8010 8011 8012, 8013, 8014, 8015, 8016 P07358 CO8G UniProtKB: 8017 8018 8019, 8020, 8021, 8022, 8023 P07360 complement_system_ UniProtKB: 8024 8025 8026, 8027, 8028, 8029, 8030 component_C1lNH P05155 complement_system_ UniProtKB: 8031 8032 8033, 8034, 8035, 8036, 8037 component_C1q11 Q8IXK1 complement_system_ UniProtKB: 8038 8039 8040, 8041, 8042, 8043, 8044 component_C1s P09871 complement_system_ UniProtKB: 8045 8046 8047, 8048, 8049, 8050, 8051 component_C4bp P04003 complement_system_ UniProtKB: 8052 8053 8054, 8055, 8056, 8057, 8058 component_C6 P13671 complement_system_ UniProtKB: 8059 8060 8061, 8062, 8063, 8064, 8065 component_C7 P10643 complement_system_ UniProtKB: 8066 8067 8068, 8069, 8070, 8071, 8072 component_C8 Q99618 complement_system_ UniProtKB: 8073 8074 8075, 8076, 8077, 8078, 8079 component_C9 A0A024R035 complement_system_ UniProtKB: 8080 8081 8082, 8083, 8084, 8085, 8086 component_C9 P02748 complement_system_ UniProtKB: 8087 8088 8089, 8090, 8091, 8092, 8093 component_CR2 P20023 complement_system_ UniProtKB: 8094 8095 8096, 8097, 8098, 8099, 8100 component_CR3 D3DSM0 complement_system_ UniProtKB: 8101 8102 8103, 8104, 8105, 8106, 8107 component_CR3 P05107 complement_system_ UniProtKB: 8108 8109 8110, 8111, 8112, 8113, 8114 component_MASP-1 P48740 complement_system_ UniProtKB: 8115 8116 8117, 8118, 8119, 8120, 8121 component_MASP-2 000187 complement_system_ UniProtKB: 8122 8123 8124, 8125, 8126, 8127, 8128 component_MBL P11226 complement_system_ UniProtKB: 8129 8130 8131, 8132, 8133, 8134, 8135 component_MCP P15529 complement_system_ UniProtKB: 8136 8137 8138, 8139, 8140, 8141, 8142 component_MCP P40121 CREB3 CCDS: 8143 8144 8145, 8146, 8147, 8148, 8149 CCDS6588.1 CREB3L1 UniProtKB: 8150 8151 8152, 8153, 8154, 8155, 8156 Q96BA8 CREB3L3 UniProtKB: 8157 8158 8159, 8160, 8161, 8162, 8163 Q68CJ9 CREB3L4 UniProtKB: 8164 8165 8166, 8167, 8168, 8169, 8170 Q8TEY5 CREBS UniProtKB: 8171 8172 8173, 8174, 8175, 8176, 8177 Q02930 CRTC2 UniProtKB: 8178 8179 8180, 8181, 8182, 8183, 8184 Q53ET0 DJB11 UniProtKB: 8185 8186 8187, 8188, 8189, 8190, 8191 Q9UBS4 DJB13 UniProtKB: 8192 8193 8194, 8195, 8196, 8197, 8198 P59910 DJB14 UniProtKB: 8199 8200 8201, 8202, 8203, 8204, 8205 Q8TBM8 DJC10 UniProtKB: 8206 8207 8208, 8209, 8210, 8211, 8212 Q81X81 DJC12 UniProtKB: 8213 8214 8215, 8216, 8217, 8218, 8219 Q9UKB3 DJC14 UniProtKB: 8220 8221 8222, 8223, 8224, 8225, 8226 Q6Y2X3 DJC15 UniProtKB: 8227 8228 8229, 8230, 8231, 8232, 8233 Q9Y5T4 DJC16 UniProtKB: 8234 8235 8236, 8237, 8238, 8239, 8240 Q9Y2G8 DJC17 UniProtKB: 8241 8242 8243, 8244, 8245, 8246, 8247 Q9NVM6 DJC18 UniProtKB: 8248 8249 8250, 8251, 8252, 8253, 8254 Q9H819 DJC22 UniProtKB: 8255 8256 8257, 8258, 8259, 8260, 8261 Q8N4W6 DJC24 UniProtKB: 8262 8263 8264, 8265, 8266, 8267, 8268 Q6P3W2 DJC25 UniProtKB: 8269 8270 8271, 8272, 8273, 8274, 8275 Q9H1X3 DJC27 UniProtKB: 8276 8277 8278, 8279, 8280, 8281, 8282 Q9NZQ0 DJC28 UniProtKB: 8283 8284 8285, 8286, 8287, 8288, 8289 Q9NX36 DJC30 UniProtKB: 8290 8291 8292, 8293, 8294, 8295, 8296 Q96LL9 DNAJB12 RefSeq: 8297 8298 8299, 8300, 8301, 8302, 8303 NP_001002762.2 DNAJC11 UniProtKB: 8304 8305 8306, 8307, 8308, 8309, 8310 Q9NVH1 DNAJC21 UniProtKB: 8311 8312 8313, 8314, 8315, 8316, 8317 Q5F1R6 DNJA1 UniProtKB: 8318 8319 8320, 8321, 8322, 8323, 8324 P31689 DNJA2 UniProtKB: 8325 8326 8327, 8328, 8329, 8330, 8331 O60884 DNJA3 UniProtKB: 8332 8333 8334, 8335, 8336, 8337, 8338 Q96EY1 DNJA4 UniProtKB: 8339 8340 8341, 8342, 8343, 8344, 8345 Q8WW22 DNJB1 UniProtKB: 8346 8347 8348, 8349, 8350, 8351, 8352 P25685 DNJB2 UniProtKB: 8353 8354 8355, 8356, 8357, 8358, 8359 P25686 DNJB3 UniProtKB: 8360 8361 8362, 8363, 8364, 8365, 8366 Q8WWF6 DNJB4 UniProtKB: 8367 8368 8369, 8370, 8371, 8372, 8373 Q9UDY4 DNJB5 UniProtKB: 8374 8375 8376, 8377, 8378, 8379, 8380 O75953 DNJB6 UniProtKB: 8381 8382 8383, 8384, 8385, 8386, 8387 O75190 DNJB7 UniProtKB: 8388 8389 8390, 8391, 8392, 8393, 8394 Q7Z6W7 DNJB8 UniProtKB: 8395 8396 8397, 8398, 8399, 8400, 8401 Q8NHS0 DNJB9 UniProtKB: 8402 8403 8404, 8405, 8406, 8407, 8408 Q9UBS3 DNJC1 UniProtKB: 8409 8410 8411, 8412, 8413, 8414, 8415 Q96KC8 DNJC2 UniProtKB: 8416 8417 8418, 8419, 8420, 8421, 8422 Q99543 DNJC3 UniProtKB: 8423 8424 8425, 8426, 8427, 8428, 8429 Q13217 DNJC4 UniProtKB: 8430 8431 8432, 8433, 8434, 8435, 8436 Q9NNZ3 DNJCS UniProtKB: 8437 8438 8439, 8440, 8441, 8442, 8443 Q9H3Z4 DNJC7 UniProtKB: 8444 8445 8446, 8447, 8448, 8449, 8450 Q99615 DNJC8 UniProtKB: 8451 8452 8453, 8454, 8455, 8456, 8457 O75937 DNJC9 UniProtKB: 8458 8459 8460, 8461, 8462, 8463, 8464 Q8WXX5 Elk-1 UniProtKB: 8465 8466 8467, 8468, 8469, 8470, 8471 P19419 Elk-1 UniProtKB: 8472 8473 8474, 8475, 8476, 8477, 8478 Q8N9S0 ERK1 UniProtKB: 8479 8480 8481, 8482, 8483, 8484, 8485 P27361 ERK2 UniProtKB: 8486 8487 8488, 8489, 8490, 8491, 8492 P28482 Fibrinogen UniProtKB: 8493 8494 8495, 8496, 8497, 8498, 8499 A0A024R8B4 Fibrinogen UniProtKB: 8500 8501 8502, 8503, 8504, 8505, 8506 A4D1B8 Fibrinogen UniProtKB: 8507 8508 8509, 8510, 8511, 8512, 8513 A8K8X4 Fibrinogen UniProtKB: 8514 8515 8516, 8517, 8518, 8519, 8520 B4DTN2 Fibrinogen UniProtKB: 8521 8522 8523, 8524, 8525, 8526, 8527 B4E1D3 Fibrinogen UniProtKB: 8528 8529 8530, 8531, 8532, 8533, 8534 D3DP13 Fibrinogen UniProtKB: 8535 8536 8537, 8538, 8539, 8540, 8541 D3DP16 Fibrinogen UniProtKB: 8542 8543 8544, 8545, 8546, 8547, 8548 D3DSP9 Fibrinogen UniProtKB: 8549 8550 8551, 8552, 8553, 8554, 8555 P02671 Fibrinogen UniProtKB: 8556 8557 8558, 8559, 8560, 8561, 8562 P02675 Fibrinogen UniProtKB: 8563 8564 8565, 8566, 8567, 8568, 8569 P02679 Fibrinogen UniProtKB: 8570 8571 8572, 8573, 8574, 8575, 8576 Q08830 Fibrinogen UniProtKB: 8577 8578 8579, 8580, 8581, 8582, 8583 Q14314 Fibrinogen UniProtKB: 8584 8585 8586, 8587, 8588, 8589, 8590 Q6UXM4 Fibrinogen UniProtKB: 8591 8592 8593, 8594, 8595, 8596, 8597 Q9UE34 FOS UniProtKB: 8598 8599 8600, 8601, 8602, 8603, 8604 A0A024RD16 GSK3A UniProtKB: 8605 8606 8607, 8608, 8609, 8610, 8611 P49840 GSK3B UniProtKB: 8612 8613 8614, 8615, 8616, 8617, 8618 P49841 HSP10 UniProtKB: 8619 8620 8621, 8622, 8623, 8624, 8625 P61604 HSP75 UniProtKB: 8626 8627 8628, 8629, 8630, 8631, 8632 Q12931 HSP90B1 UniProtKB: 8633 8634 8635, 8636, 8637, 8638, 8639 Q5CAQ5 IKK UniProtKB: 8640 8641 8642, 8643, 8644, 8645, 8646 O14920 IKK UniProtKB: 8647 8648 8649, 8650, 8651, 8652, 8653 Q14164 IKK UniProtKB: 8654 8655 8656, 8657, 8658, 8659, 8660 Q9Y6K9 IL-1 UniProtKB: 8661 8662 8663, 8664, 8665, 8666, 8667 O43353 IL-1 UniProtKB: 8668 8669 8670, 8671, 8672, 8673, 8674 Q8N9C1 IL-1 UniProtKB: 8675 8676 8677, 8678, 8679, 8680, 8681 Q8WWZ1 IL-1 UniProtKB: 8682 8683 8684, 8685, 8686, 8687, 8688 Q9NZH7 IL-1 UniProtKB: 8689 8690 8691, 8692, 8693, 8694, 8695 Q9UBH0 IL-1 UniProtKB: 8696 8697 8698, 8699, 8700, 8701, 8702 Q9UHA7 IL-1R1 UniProtKB: 8703 8704 8705, 8706, 8707, 8708, 8709 P14778 IL-1R1 UniProtKB: 8710 8711 8712, 8713, 8714, 8715, 8716 Q6NWP5 IL-1R1 UniProtKB: 8717 8718 8719, 8720, 8721, 8722, 8723 Q6NWP6 IRAK1 UniProtKB: 8724 8725 8726, 8727, 8728, 8729, 8730 L8E7M9 IRAK1 UniProtKB: 8731 8732 8733, 8734, 8735, 8736, 8737 P51617 IRAK2 UniProtKB: 8738 8739 8740, 8741, 8742, 8743, 8744 O43187 IRAK4 UniProtKB: 8745 8746 8747, 8748, 8749, 8750, 8751 Q69FE3 IRAK4 UniProtKB: 8752 8753 8754, 8755, 8756, 8757, 8758 Q7Z6A7 IRAK4 UniProtKB: 8759 8760 8761, 8762, 8763, 8764, 8765 Q7Z6A8 IRAK4 UniProtKB: 8766 8767 8768, 8769, 8770, 8771, 8772 Q9NWZ3 IRF3 UniProtKB: 8773 8774 8775, 8776, 8777, 8778, 8779 A0A024QZE1 IRF3 UniProtKB: 8780 8781 8782, 8783, 8784, 8785, 8786 E2GIM5 IRF3 UniProtKB: 8787 8788 8789, 8790, 8791, 8792, 8793 E2GIM6 IRF3 UniProtKB: 8794 8795 8796, 8797, 8798, 8799, 8800 E2GIM7 IRF3 UniProtKB: 8801 8802 8803, 8804, 8805, 8806, 8807 E2GIM8 IRF3 UniProtKB: 8808 8809 8810, 8811, 8812, 8813, 8814 E2GIM9 IRF3 UniProtKB: 8815 8816 8817, 8818, 8819, 8820, 8821 Q14653 IRF3 UniProtKB: 8822 8823 8824, 8825, 8826, 8827, 8828 Q96GL3 IRF-7 UniProtKB: 8829 8830 8831, 8832, 8833, 8834, 8835 Q92985 JNK UniProtKB: 8836 8837 8838, 8839, 8840, 8841, 8842 B4DU99 KPCB UniProtKB: 8843 8844 8845, 8846, 8847, 8848, 8849 P05771-1 KPCB UniProtKB: 8850 8851 8852, 8853, 8854, 8855, 8856 P05771-2 KPCD1 UniProtKB: 8857 8858 8859, 8860, 8861, 8862, 8863 Q15139 KPCD3 UniProtKB: 8864 8865 8866, 8867, 8868, 8869, 8870 O94806 KPCD UniProtKB: 8871 8872 8873, 8874, 8875, 8876, 8877 Q05655 KPCE UniProtKB: 8878 8879 8880, 8881, 8882, 8883, 8884 Q02156 KPCG UniProtKB: 8885 8886 8887, 8888, 8889, 8890, 8891 P05129 KPCI UniProtKB: 8892 8893 8894, 8895, 8896, 8897, 8898 P41743 KPCL UniProtKB: 8899 8900 8901, 8902, 8903, 8904, 8905 P24723 KPCT UniProtKB: 8906 8907 8908, 8909, 8910, 8911, 8912 Q04759 KPCZ UniProtKB: 8913 8914 8915, 8916, 8917, 8918, 8919 Q05513 LGP2 UniProtKB: 8920 8921 8922, 8923, 8924, 8925, 8926 A0A024R1Y5 LGP2 UniProtKB: 8927 8928 8929, 8930, 8931, 8932, 8933 Q96C10 I_IPAF UniProtKB: 8934 8935 8936, 8937, 8938, 8939, 8940 Q9NPP4 MDA5 UniProtKB: 8941 8942 8943, 8944, 8945, 8946, 8947 Q9BYX4 MK11 UniProtKB: 8948 8949 8950, 8951, 8952, 8953, 8954 Q15759 MK12 UniProtKB: 8955 8956 8957, 8958, 8959, 8960, 8961 P53778 MK13 UniProtKB: 8962 8963 8964, 8965, 8966, 8967, 8968 O15264 MK14 UniProtKB: 8969 8970 8971, 8972, 8973, 8974, 8975 Q16539 MKK1 UniProtKB: 8976 8977 8978, 8979, 8980, 8981, 8982 Q02750 MKK3 UniProtKB: 8983 8984 8985, 8986, 8987, 8988, 8989 P46734 MKK4 UniProtKB: 8990 8991 8992, 8993, 8994, 8995, 8996 P45985 MKK6 UniProtKB: 8997 8998 8999, 9000, 9001, 9002, 9003 P52564 MKK7 UniProtKB: 9004 9005 9006, 9007, 9008, 9009, 9010 O14733 MSTP104 UniProtKB: 9011 9012 9013, 9014, 9015, 9016, 9017 Q7Z4D5 MyD88 UniProtKB: 9018 9019 9020, 9021, 9022, 9023, 9024 Q99836 NALP10 UniProtKB: 9025 9026 9027, 9028, 9029, 9030, 9031 Q86W26 NALP11 UniProtKB: 9032 9033 9034, 9035, 9036, 9037, 9038 P59045 NALP12 UniProtKB: 9039 9040 9041, 9042, 9043, 9044, 9045 P59046 NALP13 UniProtKB: 9046 9047 9048, 9049, 9050, 9051, 9052 Q86W25 NALP2 UniProtKB: 9053 9054 9055, 9056, 9057, 9058, 9059 Q8WY49 NALP2 UniProtKB: 9060 9061 9062, 9063, 9064, 9065, 9066 Q9NX02 NALP3 UniProtKB: 9067 9068 9069, 9070, 9071, 9072, 9073 Q96P20 NALP4 UniProtKB: 9074 9075 9076, 9077, 9078, 9079, 9080 Q96MN2 NALP5 UniProtKB: 9081 9082 9083, 9084, 9085, 9086, 9087 P59047 NALP6 UniProtKB: 9088 9089 9090, 9091, 9092, 9093, 9094 P59044 NALP7 UniProtKB: 9095 9096 9097, 9098, 9099, 9100, 9101 Q8WX94 NALP8 UniProtKB: 9102 9103 9104, 9105, 9106, 9107, 9108 Q86W28 NALP9 UniProtKB: 9109 9110 9111, 9112, 9113, 9114, 9115 Q7RTR0 NF-kappaB UniProtKB: 9116 9117 9118, 9119, 9120, 9121, 9122 A3F768 NF-kappaB UniProtKB: 9123 9124 9125, 9126, 9127, 9128, 9129 A3F769 NLRP14 UniProtKB: 9130 9131 9132, 9133, 9134, 9135, 9136 Q86UT6 NLRP14 UniProtKB: 9137 9138 9139, 9140, 9141, 9142, 9143 Q86W24 NOD1 UniProtKB: 9144 9145 9146, 9147, 9148, 9149, 9150 G3XAL1 NOD1 UniProtKB: 9151 9152 9153, 9154, 9155, 9156, 9157 Q9Y239 NOD2 UniProtKB: 9158 9159 9160, 9161, 9162, 9163, 9164 Q9HC29 NOD3 UniProtKB: 9165 9166 9167, 9168, 9169, 9170, 9171 C3VPR7 NOD3 UniProtKB: 9172 9173 9174, 9175, 9176, 9177, 9178 H3BLT9 NOD3 UniProtKB: 9179 9180 9181, 9182, 9183, 9184, 9185 Q7RTR2 P13K UniProtKB: 9186 9187 9188, 9189, 9190, 9191, 9192 O00329 P13K UniProtKB: 9193 9194 9195, 9196, 9197, 9198, 9199 O00459 P13K UniProtKB: 9200 9201 9202, 9203, 9204, 9205, 9206 P27986 P13K UniProtKB: 9207 9208 9209, 9210, 9211, 9212, 9213 P42336 P13K UniProtKB: 9214 9215 9216, 9217, 9218, 9219, 9220 P42338 P13K UniProtKB: 9221 9222 9223, 9224, 9225, 9226, 9227 P48736 P13K UniProtKB: 9228 9229 9230, 9231, 9232, 9233, 9234 Q5UE93 P13K UniProtKB: 9235 9236 9237, 9238, 9239, 9240, 9241 Q8NEB9 P13K UniProtKB: 9242 9243 9244, 9245, 9246, 9247, 9248 Q8WYR1 PKD2 UniProtKB: 9249 9250 9251, 9252, 9253, 9254, 9255 Q13563 PKN1 UniProtKB: 9256 9257 9258, 9259, 9260, 9261, 9262 Q16512 PKN2 UniProtKB: 9263 9264 9265, 9266, 9267, 9268, 9269 Q16513 PKN3 UniProtKB: 9270 9271 9272, 9273, 9274, 9275, 9276 Q6P5Z2 PRKCA UniProtKB: 9277 9278 9279, 9280, 9281, 9282, 9283 P17252 PRKD2 RefSeq: 9284 9285 9286, 9287, 9288, 9289, 9290 NP_001073349.1 Rab UniProtKB: 9291 9292 9293, 9294, 9295, 9296, 9297 P52594 Rac1 UniProtKB: 9298 9299 9300, 9301, 9302, 9303, 9304 A4D2P0 Rac1 UniProtKB: 9305 9306 9307, 9308, 9309, 9310, 9311 A4D2P1 Rac1 UniProtKB: 9312 9313 9314, 9315, 9316, 9317, 9318 A4D2P2 Rac1 UniProtKB: 9319 9320 9321, 9322, 9323, 9324, 9325 P63000 Rac1 UniProtKB: 9326 9327 9328, 9329, 9330, 9331, 9332 WOUV93 RhoA UniProtKB: 9333 9334 9335, 9336, 9337, 9338, 9339 A0A024R324 RhoA UniProtKB: 9340 9341 9342, 9343, 9344, 9345, 9346 P61586 RIG-I UniProtKB: 9347 9348 9349, 9350, 9351, 9352, 9353 O95786 RIG-I UniProtKB: 9354 9355 9356, 9357, 9358, 9359, 9360 Q8IUD6 Src-Kinases UniProtKB: 9361 9362 9363, 9364, 9365, 9366, 9367 Q9H5V8 Surfactant_protein_A UniProtKB: 9368 9369 9370, 9371, 9372, 9373, 9374 Q8IWL1 Surfactant_protein_A UniProtKB: 9375 9376 9377, 9378, 9379, 9380, 9381 Q8IWL2 Surfactant_protein_D UniProtKB: 9382 9383 9384, 9385, 9386, 9387, 9388 P35247 TAK1 UniProtKB: 9389 9390 9391, 9392, 9393, 9394, 9395 O43318 TAK1 UniProtKB: 9396 9397 9398, 9399, 9400, 9401, 9402 P49116 TBK1 UniProtKB: 9403 9404 9405, 9406, 9407, 9408, 9409 Q9UHD2 TICAM1 UniProtKB: 9410 9411 9412, 9413, 9414, 9415, 9416 Q8IUC6 TICAM2 UniProtKB: 9417 9418 9419, 9420, 9421, 9422, 9423 Q86XR7 TIRAP UniProtKB: 9424 9425 9426, 9427, 9428, 9429, 9430 A0A024R3M4 TIRAP UniProtKB: 9431 9432 9433, 9434, 9435, 9436, 9437 P58753 TLR10 UniProtKB: 9438 9439 9440, 9441, 9442, 9443, 9444 A0A024R9W4 TLR10 UniProtKB: 9445 9446 9447, 9448, 9449, 9450, 9451 D1CS19 TLR10 UniProtKB: 9452 9453 9454, 9455, 9456, 9457, 9458 D1CS20 TLR10 UniProtKB: 9459 9460 9461, 9462, 9463, 9464, 9465 D1CS24 TLR10 UniProtKB: 9466 9467 9468, 9469, 9470, 9471, 9472 D1CS26 TLR10 UniProtKB: 9473 9474 9475, 9476, 9477, 9478, 9479 D1CS27 TLR10 UniProtKB: 9480 9481 9482, 9483, 9484, 9485, 9486 D1CS28 TLR10 UniProtKB: 9487 9488 9489, 9490, 9491, 9492, 9493 D1CS29 TLR10 UniProtKB: 9494 9495 9496, 9497, 9498, 9499, 9500 D1CS30 TLR10 UniProtKB: 9501 9502 9503, 9504, 9505, 9506, 9507 Q9BXR5 TLR1 UniProtKB: 9508 9509 9510, 9511, 9512, 9513, 9514 D1CS34 TLR1 UniProtKB: 9515 9516 9517, 9518, 9519, 9520, 9521 D1CS35 TLR1 UniProtKB: 9522 9523 9524, 9525, 9526, 9527, 9528 D1CS36 TLR1 UniProtKB: 9529 9530 9531, 9532, 9533, 9534, 9535 D1CS38 TLR1 UniProtKB: 9536 9537 9538, 9539, 9540, 9541, 9542 D1CS42 TLR1 UniProtKB: 9543 9544 9545, 9546, 9547, 9548, 9549 D1CS43 TLR1 UniProtKB: 9550 9551 9552, 9553, 9554, 9555, 9556 D1CS44 TLR1 UniProtKB: 9557 9558 9559, 9560, 9561, 9562, 9563 Q15399 TLR1 UniProtKB: 9564 9565 9566, 9567, 9568, 9569, 9570 Q5FWG5 TLR1 UniProtKB: 9571 9572 9573, 9574, 9575, 9576, 9577 Q6F164 TLR2 UniProtKB: 9578 9579 9580, 9581, 9582, 9583, 9584 O60603 TLR3 UniProtKB: 9585 9586 9587, 9588, 9589, 9590, 9591 O15455 TLR4 UniProtKB: 9592 9593 9594, 9595, 9596, 9597, 9598 D1CS55 TLR4 UniProtKB: 9599 9600 9601, 9602, 9603, 9604, 9605 O00206 TLR5 UniProtKB: 9606 9607 9608, 9609, 9610, 9611, 9612 D1CS79 TLR5 UniProtKB: 9613 9614 9615, 9616, 9617, 9618, 9619 D1CS82 TLR5 UniProtKB: 9620 9621 9622, 9623, 9624, 9625, 9626 D1CS83 TLR5 UniProtKB: 9627 9628 9629, 9630, 9631, 9632, 9633 D1CS84 TLR5 UniProtKB: 9634 9635 9636, 9637, 9638, 9639, 9640 D1CS85 TLR5 UniProtKB: 9641 9642 9643, 9644, 9645, 9646, 9647 D1CS87 TLR5 UniProtKB: 9648 9649 9650, 9651, 9652, 9653, 9654 D1CS88 TLR5 UniProtKB: 9655 9656 9657, 9658, 9659, 9660, 9661 D1CS89 TLR5 UniProtKB: 9662 9663 9664, 9665, 9666, 9667, 9668 D1CS90 TLR6 UniProtKB: 9669 9670 9671, 9672, 9673, 9674, 9675 B6CH37 TLR6 UniProtKB: 9676 9677 9678, 9679, 9680, 9681, 9682 B6CH42 TLR6 UniProtKB: 9683 9684 9685, 9686, 9687, 9688, 9689 B6CH44 TLR6 UniProtKB: 9690 9691 9692, 9693, 9694, 9695, 9696 B6CH45 TLR6 UniProtKB: 9697 9698 9699, 9700, 9701, 9702, 9703 B6RFS7 TLR6 UniProtKB: 9704 9705 9706, 9707, 9708, 9709, 9710 D1CS91 TLR6 UniProtKB: 9711 9712 9713, 9714, 9715, 9716, 9717 D1CS92 TLR6 UniProtKB: 9718 9719 9720, 9721, 9722, 9723, 9724 D1CS93 TLR6 UniProtKB: 9725 9726 9727, 9728, 9729, 9730, 9731 D1CS96 TLR6 UniProtKB: 9732 9733 9734, 9735, 9736, 9737, 9738 D1CS97 TLR6 UniProtKB: 9739 9740 9741, 9742, 9743, 9744, 9745 D1CS98 TLR6 UniProtKB: 9746 9747 9748, 9749, 9750, 9751, 9752 D1CS99 TLR6 UniProtKB: 9753 9754 9755, 9756, 9757, 9758, 9759 D1CSA0 TLR7 UniProtKB: 9760 9761 9762, 9763, 9764, 9765, 9766 B2R9N9 TLR7 UniProtKB: 9767 9768 9769, 9770, 9771, 9772, 9773 D1CS68 TLR7 UniProtKB: 9774 9775 9776, 9777, 9778, 9779, 9780 Q9NYK1 TLR8 UniProtKB: 9781 9782 9783, 9784, 9785, 9786, 9787 Q495P6 TLR8 UniProtKB: 9788 9789 9790, 9791, 9792, 9793, 9794 Q495P7 TLR8 UniProtKB: 9795 9796 9797, 9798, 9799, 9800, 9801 Q9NR97 TLR9 UniProtKB: 9802 9803 9804, 9805, 9806, 9807, 9808 B6CH46 TLR9 UniProtKB: 9809 9810 9811, 9812, 9813, 9814, 9815 D1CS61 TLR9 UniProtKB: 9816 9817 9818, 9819, 9820, 9821, 9822 D1CS62 TLR9 UniProtKB: 9823 9824 9825, 9826, 9827, 9828, 9829 L0R5D6 TLR9 UniProtKB: 9830 9831 9832, 9833, 9834, 9835, 9836 L8E8B9 TLR9 UniProtKB: 9837 9838 9839, 9840, 9841, 9842, 9843 Q9NR96 TRAF6 UniProtKB: 9844 9845 9846, 9847, 9848, 9849, 9850 Q9Y4K3 c-myc UniProtKB: 9851 9852 9853, 9854, 9855, 9856, 9857, A0A0B4J1R1 9858 c-myc UniProtKB: 9859 9860 9861, 9862, 9863, 9864, 9865, P01106 9866 c-myc UniProtKB: 9867 9868 9869, 9870, 9871, 9872, 9873, Q14901 9874 c-myc UniProtKB: 9875 9876 9877, 9878, 9879, 9880, 9881, Q16591 9882

According to the present invention, in a more preferred embodiment, the inventive composition comprises at least one RNA, preferably an mRNA comprising at least one coding region encoding at least one innate immune activator or a fragment or variant thereof, wherein the at least one coding region comprises an RNA sequence being identical or at least 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the RNA sequences according to the SEQ ID Nos as disclosed in Table 11.

13. Antibodies, Decoy Receptors and Dominant Negative Receptors:

According to a preferred embodiment the at least one RNA of the inventive RNA containing composition encodes at least one antibody and/or at least one dominant negative receptor and/or at least one decoy receptor or a fragment or variant thereof, modulating (e.g. inhibiting) the functionality of a protein or signaling pathway which is associated with tumor or cancer development. It is particularly preferred that the RNA containing composition comprises at least one RNA encoding the heavy chain of a particular antibody or fragments or variants thereof and at least one further RNA encoding the light chain of the same particular antibody or fragments or variants thereof.

In this context particularly preferred are the antibodies according to Table 12.

TABLE 12 Antibodies directed against proteins accociated with tumor or cancer development Name Target 3F8 GD2 Abagovomab CA-125 imitation Abciximab Platelet glycoprotein GPIIb/IIIa Adecatumumab EpCAM (CD326) Afutuzumab CD20 Alacizumab pegol VEGFR2 Alemtuzumab CD52 Altumomab pentetate CEA Amatuximab mesothelin Anatumomab mafenatox 5T4 Anetumab ravtansine mesothelin Apolizumab HLA-DR beta apomab TRAIL-R2 (CD262) Arcitumomab CEA Ascrinvacumab ACVRL1 Bavituximab phosphatidylserine Bectumomab CD22 Belimumab BAFF Besilesomab CEA Bevacizumab VEGF-A Bivatuzumab mertansine CD44v6 Blinatumomab CD19 × CD3 Brentuximab vedotin CD30 (TNFRSF8) Brontictuzumab NOTCH1 canakinumab IL-1β Cantuzumab mertansine CanAg Cantuzumab ravtansine MUC1 (CD227) Capromab pendetide PSMA Carlumab MCP-1 Catumaxomab EpCAM × CD3 cBR-doxorubicin immunoconjugate CD174 (Lewis Y) Cetuximab EGFR (HER1/ERBB1) Citatuzumab bogatox EpCAM Cixutumumab IGF-1R Clivatuzumab tetraxetan MUC1 (CD227) Codrituzumab glypican 3 Coltuximab ravtansine CD19 Conatumumab TRAIL-R2 (CD262) Dacetuzumab CD40 Dalotuzumab IGF-1R Dalotuzumab insulin-like growth factor I receptor Daratumumab CD38 (cyclic ADP ribose hydrolase) Demcizumab DLL4 Denintuzumab mafodotin CD19 Denosumab RANKL Depatuxizumab EGFR (HER1/ERBB1) Derlotuximab histone complex Detumomab unknown (B-lymphoma cells) Dinutuximab B4GALNT1 Drozitumab TRAIL-R2 (CD262) Duligotumab HER3 (ERBB3) Duligotuzumab EGFR (HER1/ERBB1) Dusigitumab ILGF2 Ecromeximab GD3 ganglioside Edrecolomab EpCAM Elgemtumab ERBB3 Elotuzumab SLAMF7 (CD319) Elsilimomab IL-6 Emactuzumab CSF1R Emibetuzumab HGFR Emibetuzumab MET Enavatuzumab TNFRSF12A Enfortumab vedotin AGS-22M6 Enoticumab DLL4 Ensituximab MUC5AC Epitumomab cituxetan MUC1 (CD227) Epratuzumab CD22 Ertumaxomab HER2 (ERBB2/neu) × CD3 Etaracizumab integrin α5β3 Faralimomab IFNA1 Farletuzumab FOLR1 alpha FBTA CD20 × CD3 Ficlatuzumab HGFR Figitumumab IGF-1R Flanvotumab TYRP1(glycoprotein 75) Fresolimumab TGF-β Futuximab EGFR (HER1/ERBB1) Galiximab CD80 Gantiumab IGF-1R Gemtuzumab ozogamicin CD33 Girentuximab Carbonic anhydrase 9 (CA9/CAIX) Glembatumumab vedotin GPNMB glycooptimized trastuzumab-GEX HER2 (ERBB2/neu) Ibritumomab tiuxetan CD20 Icrucumab VEGFR-1 Igovomab MUC16 IMAB362 Claudin-18 (CLDN18.2) Imgatuzumab EGFR (HER1/ERBB1) Indatuximab ravtansine SDC1 Indusatumab vedotin GUCY2C inebilizumab CD19 Inotuzumab ozogamicin CD22 Intetumumab CD51 Iratumumab CD30 (TNFRSF8) Isatuximab CD38 Labetuzumab CEA Lenzilumab CSF2 Lexatumumab TRAIL-R2 (CD262) Lifastuzumab vedotin NaPi2B Lilotomab satetraxetan CD37 Lintuzumab CD33 Lorvotuzumab mertansine CD56 Lucatumumab CD40 Lumiliximab CD23 (IgE receptor) Lumretuzumab ERBB3 Mapatumumab TRAIL-R1 (CD261) Margetuximab HER2 (ERBB2/neu) Matuzumab EGFR (HER1/ERBB1) Mepolizumab IL-5 Milatuzumab CD74 Minretumomab TAG-72 Mirvetuximab soravtansine FOLR1 alpha Mitumomab GD3 (ganglioside) Mogamulizumab CCR4 Moxetumomab pasudotox CD22 Nacolomab tafenatox C242 antigen Naptumomab estafenatox 5T4 Narnatumab RON Necitumumab EGFR (HER1/ERBB1) Nesvacumab ANGPT2 (angiopoietin 2) Nimotuzumab EGFR (HER1/ERBB1) Nofetumomab merpentan EpCAM binutuzumab CD20 Ocaratuzumab CD20 Ofatumumab CD20 Olaratumab PDGFRα Onartuzumab MET Ontuxizumab CD248 (TEM1) Oportuzumab monatox EpCAM Oregovomab CA-125 Otlertuzumab CD37 Panitumumab EGFR (HER1/ERBB1) Pankomab MUC1 (tumor specific glycosylation) Parsatuzumab EGFL7 Pasotuxizumab FOLH1 Patritumab HER3 (ERBB3) Pemtumomab MUC1 (CD227) Pertuzumab HER2 (ERBB2/neu) Pinatuzumab vedotin CD22 Pintumomab adenocarcinoma antigen Polatuzumab vedotin CD79B Racotumomab NGcGM3 Radretumab EDB (fibronectin extra domain-B) Ramucirumab VEGFR2 Rilotumumab HGFR Rituximab CD20 Robatumumab IGF-1R Sacituzumab govitecan Trop-2 (tumor-associated calcium signal transducer 2/EGP-1) Samalizumab CD200 (OX-2 membrane glycoprotein) Satumomab pendetide TAG-72 Seribantumab ERBB3 Seribantumab HER3 (ERBB3) SGN-CDA CD19 SGN-CDA CD33 Sibrotuzumab FAP Siltuximab IL-6 Simtuzumab LOXL2 Sofituzumab vedotin CA 125 Solitomab EpCAM Sonepcizumab S1P (sphingosine-1-phosphate) Tacatuzumab tetraxetan AFP (alpha-fetoprotein) Taplitumomab paptox CD19 Tarextumab Notch receptor Tenatumomab TN-C (tenascin C) Teprotumumab CD221 Tetulomab CD37 TGN CD28 Tigatuzumab TRAIL-R2 (CD262) Lebrikizumab IL-13 Tocilizumab IL-6R Tositumomab CD20 Tovetumab CD140a Tovetumab PDGFRα Trastuzumab HER2 (ERBB2/neu) Trastuzumab emtansine HER2 (ERBB2/neu) TRBS GD2 Tucotuzumab celmoleukin EpCAM ublituximab CD20 Ublituximab MS4A1 Ulocuplumab CXCR4 Vandortuzumab vedotin STEAP1 Vantictumab FZD7 Vanucizumab Ang-2 (angiopoietin 2) × VEGF-A Veltuzumab CD20 Vesencumab NRP1 Volociximab integrin α5β1 Votumumab CTAA16.88 Zalutumumab EGFR (HER1/ERBB1) Zanolimumab CD4 Zatuximab HER1 (EGFR/ERBB1)

Preferably, the neutralizing antibody is chosen from the list of anti-IL-10 and anti-TGFbeta.

Furthermore, the at least one antibody may preferably chosen from anti-CD73 antibodies or fragments or variants thereof.

In a further particularly preferred embodiment the at least one antibody is chosen from an antibody directed against CCR5/CD195 or from an antibody directed against its ligand CCL5/RANTES.

In a particularly preferred embodiment the decoy receptor is a soluble CCR5 (chemokine receptor type 5, also known as CD195).

In a further particularly preferred embodiment the dominant negative receptor is dominant negative CCR5 (chemokine receptor type 5, also known as CD195).

Furthermore, the at least one antibody may preferably chosen from anti-CD73 antibodies or fragments or variants thereof.

14. Inhibitors of Myeloid Derived Suppressor Cells (MDSCs):

Myeloid Derived Suppressor Cells (MDSC) are a heterogeneous population of immature myeloid cells that are increased in cancer and related disorders. MDSC are induced by tumor secreted growth factors. MDSC play an important part in suppression of host immune responses through several mechanisms. In addition, MDSC may also contribute to angiogenesis and tumor invasion. Therefore, MDSC inhibition is a strategy for the treatment of cancer and related disorders.

In the context of the invention, MDSC inhibition can be achieved by direct deactivation of MDSCs (e.g., anti IL-17 antibodies), by blocking differentiation of MDSCs into mature cells (e.g., IL-12), by blocking the cell development of MDSCs or by depletion of MDSCs (e.g., cytotoxic agents).

Therefore it is particularly preferred to use anti IL-17 antibodies and IL-12 as inhibitors of MDSCs.

15. IDO Pathway Inhibitors

In a further preferred embodiment of the inventive RNA containing composition the RNA, preferably mRNA codes for at least one IDO pathway inhibitor. Preferably the RNA encoding the at least one IDO pathway inhibitor encodes an inhibitory protein or dominant negative mutant protein of the IDO pathway.

As reviewed in Prendergast et al. (Prendergast G C, Smith C, Thomas S, Mandik-Nayak L, Laury-Kleintop L, Metz R, Muller A J. Indoleamine 2,3-dioxygenase pathways of pathogenic inflammation and immune escape in cancer. Cancer Immunol. Immunother. 2014 July; 63(7):721-35) indoleamine-pyrrole 2,3-dioxygenase (IDO or INDO EC 1.13.11.52) is an enzyme that in humans is encoded by the IDO1 gene. This enzyme catalyzes the degradation of the essential amino acid L-tryptophan to N-formylkynurenine. IDO is the first and rate-limiting enzyme of tryptophan catabolism through kynurenine pathway, thus causing depletion of tryptophan which can cause halted growth of microbes as well as T cells. IDO is an immunomodulatory enzyme produced by some alternatively activated macrophages and other immunoregulatory cells (also used as an immune subversion strategy by many tumors). The clinical development of IDO inhibitors may produce a novel class of immunomodulators with broad application in the treatment of advanced human cancer.

16. Proteins or Peptides that Bind Inhibitors of Apoptosis

Apoptosis is a tightly regulated cellular process and faulty regulation of apoptosis is a hallmark of human cancers. Targeting key apoptosis regulators with the goal to restore apoptosis in tumor cells has been pursued as a new cancer therapeutic strategy. XIAP, cIAP1, and cIAP2, members of inhibitor of apoptosis (IAP) proteins, are critical regulators of cell death and survival and are attractive targets for new cancer therapy. The SMAC/DIABLO protein is an endogenous antagonist of XIAP, cIAP1, and cIAP2. In the last decade, intense research efforts have resulted in the design and development of several small-molecule SMAC mimetics now in clinical trials for cancer treatment

In a further preferred embodiment, the inventive composition comprises at least one RNA comprising at least one coding region that codes for at least one peptide or protein that binds inhibitors of apoptosis proteins (IAPB) and thus sensitize cancer cells to apoptotic death.

Therefore it is particularly preferred that the at least one RNA of the inventive RNA containing composition encodes at least one protein or peptide that bind inhibitors of apoptosis, such as SMAC mimetics.

Particularly preferred proteins or peptides that bind IAPB according to the present invention comprise Omi/HtrA2, Smac, Smac derived peptides, Smac/DIABLO, and XAF1 (XIAP-associated factor 1) and fragments or variants thereof.

RNA Modifications

According to one embodiment, the at least one RNA of the composition, encoding at least one of the proteins and/or peptides defined herein, may be in the form of a modified RNA, wherein any modification, as defined herein, may be introduced into the at least one RNA of the composition. Modifications as defined herein preferably lead to a stabilization of the at least one RNA of the composition of the present invention.

According to one embodiment, the at least one RNA of the composition of the present invention may thus be provided as a “stabilized RNA”, that is to say as an RNA that is essentially resistant to in vivo degradation (e.g. by an exo- or endo-nuclease). Such stabilization can be effected, for example, by a modified phosphate backbone of the at least one RNA of the composition of the present invention. A backbone modification in connection with the present invention is a modification in which phosphates of the backbone of the nucleotides contained in the RNA are chemically modified. Nucleotides that may be preferably used in this connection contain e.g. a phosphorothioate-modified phosphate backbone, preferably at least one of the phosphate oxygens contained in the phosphate backbone being replaced by a sulfur atom. Stabilized RNAs may further include, for example: non-ionic phosphate analogues, such as, for example, alkyl and aryl phosphonates, in which the charged phosphonate oxygen is replaced by an alkyl or aryl group, or phosphodiesters and alkylphosphotriesters, in which the charged oxygen residue is present in alkylated form. Such backbone modifications typically include, without implying any limitation, modifications from the group consisting of methylphosphonates, phosphoramidates and phosphorothioates (e.g. cytidine-5′-O-(1-thiophosphate)).

In the following, specific modifications are described, which are preferably capable of “stabilizing” the at least one RNA as defined herein.

Chemical Modifications:

The term “RNA modification” as used herein may refer to chemical modifications comprising backbone modifications as well as sugar modifications or base modifications.

In this context, a modified RNA as defined herein may contain nucleotide analogues/modifications, e.g. backbone modifications, sugar modifications or base modifications. A backbone modification in connection with the present invention is a modification, in which phosphates of the backbone of the nucleotides contained in an RNA as defined herein are chemically modified. A sugar modification in connection with the present invention is a chemical modification of the sugar of the nucleotides of the RNA as defined herein. Furthermore, a base modification in connection with the present invention is a chemical modification of the base moiety of the nucleotides of the RNA. In this context, nucleotide analogues or modifications are preferably selected from nucleotide analogues, which are applicable for transcription and/or translation.

Sugar Modifications:

The modified nucleosides and nucleotides, which may be incorporated into a modified RNA as described herein, can be modified in the sugar moiety. For example, the 2′ hydroxyl group (OH) can be modified or replaced with a number of different “oxy” or “deoxy” substituents. Examples of “oxy”-2′ hydroxyl group modifications include, but are not limited to, alkoxy or aryloxy (—OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG), —O(CH₂CH₂O)nCH₂CH₂OR; “locked” nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by a methylene bridge, to the 4′ carbon of the same ribose sugar; and amino groups (—O-amino, wherein the amino group, e.g., NRR, can be alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroaryl amino, ethylene diamine, polyamino) or aminoalkoxy.

“Deoxy” modifications include hydrogen, amino (e.g. NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); or the amino group can be attached to the sugar through a linker, wherein the linker comprises one or more of the atoms C, N, and O.

The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified RNA can include nucleotides containing, for instance, arabinose as the sugar.

Backbone Modifications:

The phosphate backbone may further be modified in the modified nucleosides and nucleotides, which may be incorporated into a modified RNA as described herein. The phosphate groups of the backbone can be modified by replacing one or more of the oxygen atoms with a different substituent. Further, the modified nucleosides and nucleotides can include the full replacement of an unmodified phosphate moiety with a modified phosphate as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters.

Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylene-phosphonates).

Base Modifications:

The modified nucleosides and nucleotides, which may be incorporated into a modified RNA as described herein can further be modified in the nucleobase moiety. Examples of nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine and uracil. For example, the nucleosides and nucleotides described herein can be chemically modified on the major groove face. In some embodiments, the major groove chemical modifications can include an amino group, a thiol group, an alkyl group, or a halo group.

In particularly preferred embodiments of the present invention, the nucleotide analogues/modifications are selected from base modifications, which are preferably selected from 2-amino-6-chloropurineriboside-5′-triphosphate, 2-Aminopurine-riboside-5′-triphosphate; 2-aminoadenosine-5′-triphosphate, 2′-Amino-2′-deoxycytidine-triphosphate, 2-thiocytidine-5′-triphosphate, 2-thiouridine-5′-triphosphate, 2′-Fluorothymidine-5′-triphosphate, 2′-O-Methyl inosine-5′-triphosphate 4-thiouridine-5′-triphosphate, 5-aminoallylcytidine-5′-triphosphate, 5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5′-triphosphate, 5-bromouridine-5′-triphosphate, 5-Bromo-2′-deoxycytidine-5′-triphosphate, 5-Bromo-2′-deoxyuridine-5′-triphosphate, 5-iodocytidine-5′-triphosphate, 5-Iodo-2′-deoxycytidine-5′-triphosphate, 5-iodouridine-5′-triphosphate, 5-Iodo-2′-deoxyuridine-5′-triphosphate, 5-methylcytidine-5′-triphosphate, 5-methyluridine-5′-triphosphate, 5-Propynyl-2′-deoxycytidine-5′-triphosphate, 5-Propynyl-2′-deoxyuridine-5′-triphosphate, 6-azacytidine-5′-triphosphate, 6-azauridine-5′-triphosphate, 6-chloropurineriboside-5′-triphosphate, 7-deazaadenosine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate, 8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate, benzimidazole-riboside-5′-triphosphate, N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate, N6-methyladenosine-5′-triphosphate, 06-methylguanosine-5′-triphosphate, pseudouridine-5′-triphosphate, or puromycin-5′-triphosphate, xanthosine-5′-triphosphate. Particular preference is given to nucleotides for base modifications selected from the group of base-modified nucleotides consisting of 5-methylcytidine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate, 5-bromocytidine-5′-triphosphate, and pseudouridine-5′-triphosphate.

In some embodiments, modified nucleosides include pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, I-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-I-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.

In some embodiments, modified nucleosides include 5-aza-cytidine, pseudoisocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methylcytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methylcytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

In other embodiments, modified nucleosides include 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

In other embodiments, modified nucleosides include inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, I-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

In some embodiments, the nucleotide can be modified on the major groove face and can include replacing hydrogen on C-5 of uracil with a methyl group or a halo group.

In specific embodiments, a modified nucleoside is 5′-O-(1-thiophosphate)-adenosine, 5′-O-(1-thiophosphate)-cytidine, 5′-O-(1-thiophosphate)-guanosine, 5′-O-(1-thiophosphate)-uridine or 5′-O-(1-thiophosphate)-pseudouridine.

In further specific embodiments, a modified RNA may comprise nucleoside modifications selected from 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, Pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodouridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxyuridine, deoxy-thymidine, 5-methyl-uridine, Pyrrolo-cytidine, inosine, α-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytdine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-Chloro-purine, N6-methyl-2-amino-purine, Pseudo-iso-cytidine, 6-Chloro-purine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine.

Lipid Modification:

According to a further embodiment, a modified RNA as defined herein can contain a lipid modification. Such a lipid-modified RNA typically comprises an RNA as defined herein. Such a lipid-modified RNA as defined herein typically further comprises at least one linker covalently linked with that RNA, and at least one lipid covalently linked with the respective linker. Alternatively, the lipid-modified RNA comprises at least one RNA as defined herein and at least one (bifunctional) lipid covalently linked (without a linker) with that RNA. According to a third alternative, the lipid-modified RNA comprises an RNA molecule as defined herein, at least one linker covalently linked with that RNA, and at least one lipid covalently linked with the respective linker, and also at least one (bifunctional) lipid covalently linked (without a linker) with that RNA. In this context, it is particularly preferred that the lipid modification is present at the terminal ends of a linear RNA sequence.

G/C Content Optimization:

According to an especially preferred embodiment of the invention, the RNA of the inventive composition is modified. Preferably the RNA is stabilized by modifying and preferably increasing the G (guanosine)/C (cytosine) content of the RNA of the coding region thereof. Therein, the G/C content of the RNA of the coding region is increased compared to the G/C content of the coding region of its particular wild type coding sequence, i.e. the unmodified RNA. However, the encoded amino acid sequence of the RNA is preferably not modified compared to the encoded amino acid sequence of the particular wild type/unmodified RNA.

The modification of the G/C-content of the RNA of the inventive composition is based on the fact that RNA sequences having an increased G (guanosine)/C (cytosine) content are more stable than RNA sequences having an increased A (adenosine)/U (uracil) content. The codons of a coding sequence or a whole RNA might therefore be varied compared to the wild type coding sequence or RNA, such that they include an increased amount of G/C nucleotides while the translated amino acid sequence is retained. In respect to the fact that several codons code for one and the same amino acid (so-called degeneration of the genetic code), the most favourable codons for the stability can be determined (so-called alternative codon usage). Depending on the amino acid to be encoded by the at least one RNA, there are various possibilities for modification of the RNA sequence, compared to its wild-type sequence. In the case of amino acids which are encoded by codons, which contain exclusively G or C nucleotides, no modification of the codon is necessary. Thus, the codons for Pro (CCC or CCG), Arg (CGC or CGG), Ala (GCC or GCG) and Gly (GGC or GGG) require no modification, since no A or U is present. In contrast, codons which contain A and/or U nucleotides can be modified by substitution of other codons, which code for the same amino acids but contain no A and/or U. Examples of these are: the codons for Pro can be modified from CCU or CCA to CCC or CCG; the codons for Arg can be modified from CGU or CGA or AGA or AGG to CGC or CGG; the codons for Ala can be modified from GCU or GCA to GCC or GCG; the codons for Gly can be modified from GGU or GGA to GGC or GGG. In other cases, although A or U nucleotides cannot be eliminated from the codons, it is however possible to decrease the A and U content by using codons which contain a lower content of A and/or U nucleotides. Examples of these are: the codons for Phe can be modified from UUU to UUC; the codons for Leu can be modified from UUA, UUG, CUU or CUA to CUC or CUG; the codons for Ser can be modified from UCU or UCA or AGU to UCC, UCG or AGC; the codon for Tyr can be modified from UAU to UAC; the codon for Cys can be modified from UGU to UGC; the codon for His can be modified from CAU to CAC; the codon for Gln can be modified from CAA to CAG; the codons for Ile can be modified from AUU or AUA to AUC; the codons for Thr can be modified from ACU or ACA to ACC or ACG; the codon for Asn can be modified from AAU to AAC; the codon for Lys can be modified from AAA to AAG; the codons for Val can be modified from GUU or GUA to GUC or GUG; the codon for Asp can be modified from GAU to GAC; the codon for Glu can be modified from GAA to GAG; the stop codon UAA can be modified to UAG or UGA. In the case of the codons for Met (AUG) and Trp (UGG), on the other hand, there is no possibility of sequence modification. The substitutions listed above can be used either individually or in all possible combinations to increase the G/C content of the at least one mRNA of the composition of the present invention compared to its particular wild-type mRNA (i.e. the original sequence). Thus, for example, all codons for Thr occurring in the wild-type sequence can be modified to ACC (or ACG). Preferably, however, for example, combinations of the above substitution possibilities are used:

substitution of all codons coding for Thr in the original sequence (wild-type mRNA) to ACC (or ACG) and substitution of all codons originally coding for Ser to UCC (or UCG or AGC); substitution of all codons coding for Ile in the original sequence to AUC and

substitution of all codons originally coding for Lys to AAG and

substitution of all codons originally coding for Tyr to UAC; substitution of all codons coding for Val in the original sequence to GUC (or GUG) and

substitution of all codons originally coding for Glu to GAG and

substitution of all codons originally coding for Ala to GCC (or GCG) and

substitution of all codons originally coding for Arg to CGC (or CGG); substitution of all codons coding for Val in the original sequence to GUC (or GUG) and

substitution of all codons originally coding for Glu to GAG and

substitution of all codons originally coding for Ala to GCC (or GCG) and

substitution of all codons originally coding for Gly to GGC (or GGG) and

substitution of all codons originally coding for Asn to AAC; substitution of all codons coding for Val in the original sequence to GUC (or GUG) and

substitution of all codons originally coding for Phe to UUC and

substitution of all codons originally coding for Cys to UGC and

substitution of all codons originally coding for Leu to CUG (or CUC) and

substitution of all codons originally coding for Gln to CAG and

substitution of all codons originally coding for Pro to CCC (or CCG); etc.

Preferably, the G/C content of the coding region of the at least one RNA according to the invention is increased by at least 7%, more preferably by at least 15%, particularly preferably by at least 20%, compared to the G/C content of the coding region of the wild type RNA. According to a specific embodiment at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, more preferably at least 70%, even more preferably at least 80% and most preferably at least 90%, 95% or even 100% of the substitutable codons in the region coding for a protein or peptide as defined herein or its fragment or variant thereof or the whole sequence of the wild type RNA sequence or coding sequence are substituted, thereby increasing the G/C content of said sequence. In this context, it is particularly preferable to increase the G/C content of the at least one RNA of the inventive composition to the maximum (i.e. 100% of the substitutable codons), in particular in the coding region, compared to the wild type sequence.

According to the invention, a further preferred modification of the coding sequence of the at least one RNA of the composition is based on the finding that the translation efficiency is also determined by a different frequency in the occurrence of tRNAs in cells. Thus, if so-called “rare codons” are present in the at least one coding region of the at least one RNA of the composition of the present invention to an increased extent, the corresponding modified at least one RNA sequence is translated to a significantly poorer degree than in the case where codons coding for relatively “frequent” tRNAs are present. According to the invention, in the modified at least one RNA of the composition of the present invention, the region which codes for one of the above defined peptides or proteins is modified compared to the corresponding region of the wild-type RNA such that at least one codon of the wild-type sequence, which codes for a tRNA which is relatively rare in the cell, is exchanged for a codon, which codes for a tRNA which is relatively frequent in the cell and carries the same amino acid as the relatively rare tRNA. By this modification, the sequence of the at least one coding region of the at least one RNA of the composition of the present invention is modified such that codons for which frequently occurring tRNAs are available are inserted. In other words, according to the invention, by this modification all codons of the wild-type sequence which code for a tRNA which is relatively rare in the cell can in each case be exchanged for a codon which codes for a tRNA which is relatively frequent in the cell and which, in each case, carries the same amino acid as the relatively rare tRNA. Which tRNAs occur relatively frequently in the cell and which, in contrast, occur relatively rarely is known to a person skilled in the art; cf. e.g. Akashi, Curr. Opin. Genet. Dev. 2001, 11(6): 660-666. The codons which use for the particular amino acid the tRNA which occurs the most frequently, e.g. the Gly codon, which uses the tRNA, which occurs the most frequently in the (human) cell, are particularly preferred. According to the invention, it is particularly preferable to link the sequential G/C content which is increased, in particular maximized, in the modified at least one RNA of the composition of the present invention, with the “frequent” codons without modifying the amino acid sequence of the protein encoded by the coding region of the RNA. This preferred embodiment allows provision of a particularly efficiently translated and stabilized (modified) at least one RNA of the composition of the present invention. The determination of a modified at least one RNA of the composition of the present invention as described above (increased G/C content; exchange of tRNAs) can be carried out using the computer program explained in WO 02/098443—the disclosure content of which is included in its full scope in the present invention. Using this computer program, the nucleotide sequence of any desired coding RNA can be modified with the aid of the genetic code or the degenerative nature thereof such that a maximum G/C content results, in combination with the use of codons which code for tRNAs occurring as frequently as possible in the cell, the amino acid sequence coded by the modified at least one RNA preferably not being modified compared to the non-modified sequence. Alternatively, it is also possible to modify only the G/C content or only the codon usage compared to the original sequence. The source code in Visual Basic 6.0 (development environment used: Microsoft Visual Studio Enterprise 6.0 with Servicepack 3) is also described in WO 02/098443. In a further preferred embodiment of the present invention, the A/U content in the environment of the ribosome binding site of the at least one RNA of the composition of the present invention is increased compared to the A/U content in the environment of the ribosome binding site of its particular wild-type RNA. This modification (an increased A/U content around the ribosome binding site) increases the efficiency of ribosome binding to the at least one RNA. An effective binding of the ribosomes to the ribosome binding site (Kozak sequence: GCCGCCACCAUGG (SEQ ID NO: 10.071), the AUG forms the start codon) in turn has the effect of an efficient translation of the at least one RNA. According to a further embodiment of the present invention the at least one RNA of the composition of the present invention may be modified with respect to potentially destabilizing sequence elements. Particularly, the coding region and/or the 5′ and/or 3′ untranslated region of this RNA may be modified compared to the particular wild-type RNA such that it contains no destabilizing sequence elements, the coded amino acid sequence of the modified at least one RNA preferably not being modified compared to its particular wild-type RNA. It is known that, for example, in sequences of eukaryotic RNAs destabilizing sequence elements (DSE) occur, to which signal proteins bind and regulate enzymatic degradation of RNA in vivo. For further stabilization of the modified at least one RNA, optionally in the region which encodes for a protein or peptide as defined herein, one or more such modifications compared to the corresponding region of the wild-type RNA can therefore be carried out, so that no or substantially no destabilizing sequence elements are contained there. According to the invention, DSE present in the untranslated regions (3′- and/or 5′-UTR) can also be eliminated from the at least one RNA of the composition of the present invention by such modifications. Such destabilizing sequences are e.g. AU-rich sequences (AURES), which occur in 3′-UTR sections of numerous unstable RNAs (Caput et al., Proc. Natl. Acad. Sci. USA 1986, 83: 1670 to 1674). The at least one RNA of the composition of the present invention is therefore preferably modified compared to the wild-type RNA such that the at least one RNA contains no such destabilizing sequences. This also applies to those sequence motifs which are recognized by possible endonucleases, e.g. the sequence GAACAAG, which is contained in the 3′-UTR segment of the gene which codes for the transferrin receptor (Binder et al., EMBO J. 1994, 13: 1969 to 1980). These sequence motifs are also preferably removed in the at least one RNA of the composition of the present invention.

Adaptation to Human Codon Usage:

According to the invention, a further preferred modification of the at least one RNA of the composition of the present invention is based on the finding that codons coding for the same amino acid occur in different frequencies. According to the invention, in the modified at least one RNA of the composition of the present invention, the region which codes for one of the above defined peptides or proteins (coding sequence) is preferably modified compared to the corresponding region of the wild-type RNA such that the frequency of the codons coding for the same amino acid corresponds to the naturally occurring frequency of that codon present in the human coding usage as e.g. shown in Table 13.

This means, for example, that for the amino acid Alanine (Ala) present in the amino acid sequence of the encoded protein according to the invention, the wild type coding sequence is adapted in a way that the codon “GCC” is used with a frequency of 0.40, the codon “GCT” is used with a frequency of 0.28, the codon “GCA” is used with a frequency of 0.22 and the codon “GCG” is used with a frequency of 0.10 etc. (see Table 13).

TABLE 13 Human codon usage table (most frequent codon marked with an asterisk) Amino acid codon fraction /1000 Ala GCG 0.10 7.4 Ala GCA 0.22 15.8 Ala GCT 0.28 18.5 Ala  GCC* 0.40 27.7 Cys TGT 0.42 10.6 Cys  TGC* 0.58 12.6 Asp GAT 0.44 21.8 Asp  GAC* 0.56 25.1 Glu  GAG* 0.59 39.6 Glu GAA 0.41 29.0 Phe TTT 0.43 17.6 Phe  TTC* 0.57 20.3 Gly GGG 0.23 16.5 Gly GGA 0.26 16.5 Gly GGT 0.18 10.8 Gly  GGC* 0.33 22.2 His CAT 0.41 10.9 His  CAC* 0.59 15.1 Ile ATA 0.14 7.5 Ile ATT 0.35 16.0 Ile  ATC* 0.52 20.8 Lys  AAG* 0.60 31.9 Lys AAA 0.40 24.4 Leu TTG 0.12 12.9 Leu TTA 0.06 7.7 Leu  CTG* 0.43 39.6 Leu CTA 0.07 7.2 Leu CTT 0.12 13.2 Leu CTC 0.20 19.6 Met  ATG* 1 22.0 Asn AAT 0.44 17.0 Asn  AAC* 0.56 19.1 Pro CCG 0.11 6.9 Pro CCA 0.27 16.9 Pro CCT 0.29 17.5 Pro  CCC* 0.33 19.8 Gln  CAG* 0.73 34.2 Gln CAA 0.27 12.3 Arg AGG 0.22 12.0 Arg  AGA* 0.21 12.1 Arg CGG 0.19 11.4 Arg CGA 0.10 6.2 Arg CGT 0.09 4.5 Arg CGC 0.19 10.4 Ser AGT 0.14 12.1 Ser  AGC* 0.25 19.5 Ser TCG 0.06 4.4 Ser TCA 0.15 12.2 Ser TCT 0.18 15.2 Ser TCC 0.23 17.7 Thr ACG 0.12 6.1 Thr ACA 0.27 15.1 Thr ACT 0.23 13.1 Thr  ACC* 0.38 18.9 Val  GTG* 0.48 28.1 Val GTA 0.10 7.1 Val GTT 0.17 11.0 Val GTC 0.25 14.5 Trp  TGG* 1 13.2 Tyr TAT 0.42 12.2 Tyr  TAC* 0.58 15.3 Stop  TGA* 0.61 1.6 Stop TAG 0.17 0.8 Stop TAA 0.22 1.0

Codon-Optimization:

According to a particularly preferred embodiment it is preferred, that all codons of the wild-type sequence of the coding region of the at least one RNA of the inventive composition which code for a tRNA which is relatively rare in the cell is in each case exchanged for a codon which codes for a tRNA which is relatively frequent in the cell and which, in each case, carries the same amino acid as the relatively rare tRNA. Therefore it is particularly preferred that the most frequent codons are used for each encoded amino acid (see Table 13, most frequent codons are marked with asterisks).

This means, for example, that for the amino acid Alanine (Ala) present in the amino acid sequence of the encoded peptide or protein according to the invention, the wild type coding sequence is adapted in a way that the most frequent human codon “GCC” is always used for said amino acid, or for the amino acid Cysteine (Cys), the wild type sequence is adapted in a way that the most frequent human codon “TGC” is always used for said amino acid etc.

C-Enrichment:

According to another embodiment, the at least one RNA of the composition of the present invention may be modified by increasing the C content of the RNA, preferably of the coding region of the at least one RNA.

In a particularly preferred embodiment of the present invention, the C content of the coding region of the at least one RNA of the composition of the present invention is modified, particularly increased, compared to the C content of the coding region of its particular wild-type RNA, i.e. the unmodified mRNA. The amino acid sequence encoded by the at least one RNA is preferably not modified as compared to the amino acid sequence encoded by the particular wild-type RNA

In a preferred embodiment of the present invention, the modified RNA is modified such that at least 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%, or at least 90% of the theoretically maximal cytosine-content or even a maximal cytosine-content is achieved.

In further preferred embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even 100% of the codons of the target RNA wild type sequence, which are “cytosine content optimizable” are replaced by codons with a higher cytosine-content as present in the wild type sequence.

In a further preferred embodiment, some of the codons of the wild type coding sequence may additionally be modified such that a codon for a relatively rare tRNA in the cell is exchanged by a codon for a relatively frequent tRNA in the cell, provided that the substituted codon for a relatively frequent tRNA carries the same amino acid as the relatively rare tRNA of the original wild type codon. Preferably, all of the codons for a relatively rare tRNA are replaced by a codon for a relatively frequent tRNA in the cell, except codons encoding amino acids, which are exclusively encoded by codons not containing any cytosine, or except for glutamine (Gln), which is encoded by two codons each containing the same number of cytosines.

In a further preferred embodiment of the present invention, the modified target RNA is modified such that at least 80%, or at least 90% of the theoretically maximal cytosine-content or even a maximal cytosine-content is achieved by means of codons, which code for relatively frequent tRNAs in the cell, wherein the amino acid sequence remains unchanged.

Due to the naturally occurring degeneracy of the genetic code, more than one codon may encode a particular amino acid. Accordingly, 18 out of 20 naturally occurring amino acids are encoded by more than 1 codon (with Tryp and Met being an exception), e.g. by 2 codons (e.g. Cys, Asp, Glu), by three codons (e.g. Ile), by 4 codons (e.g. Al, Gly, Pro) or by 6 codons (e.g. Leu, Arg, Ser). However, not all codons encoding the same amino acid are utilized equally frequent under in vivo conditions. Depending on each single organism, a typical codon usage profile is established.

The term “cytosine content-optimizable codon” as used within the context of the present invention refers to codons, which exhibit a lower amount of cytosines than other codons coding for the same amino acid. Accordingly, any wild type codon, which may be replaced by another codon coding for the same amino acid and exhibiting a higher number of cytosines within that codon, is considered to be cytosine-optimizable (C-optimizable). Any such substitution of a C-optimizable wild type codon by the specific C-optimized codon within a wild type coding region increases its overall C-content and reflects a C-enriched modified RNA sequence. A C-maximized RNA sequence contains C-optimized codons for all potentially C-optimizable codons. Accordingly, 100% or all of the theoretically replaceable C-optimizable codons are under such conditions actually replaced by C-optimized codons over the entire length of the coding region.

In this context, cytosine-content optimizable codons are codons, which contain a lower number of cytosines than other codons coding for the same amino acid.

Any of the codons GCG, GCA, GCU codes for the amino acid Ala, which may be exchanged by the codon GCC encoding the same amino acid, and/or

the codon UGU that codes for Cys may be exchanged by the codon UGC encoding the same amino acid, and/or

the codon GAU which codes for Asp may be exchanged by the codon GAC encoding the same amino acid, and/or

the codon that UUU that codes for Phe may be exchanged for the codon UUC encoding the same amino acid, and/or

any of the codons GGG, GGA, GGU that code Gly may be exchanged by the codon GGC encoding the same amino acid, and/or

the codon CAU that codes for His may be exchanged by the codon CAC encoding the same amino acid, and/or

any of the codons AUA, AUU that code for Ile may be exchanged by the codon AUC, and/or

any of the codons UUG, UUA, CUG, CUA, CUU coding for Leu may be exchanged by the codon CUC encoding the same amino acid, and/or

the codon AAU that codes for Asn may be exchanged by the codon AAC encoding the same amino acid, and/or

any of the codons CCG, CCA, CCU coding for Pro may be exchanged by the codon CCC encoding the same amino acid, and/or

any of the codons AGG, AGA, CGG, CGA, CGU coding for Arg may be exchanged by the codon CGC encoding the same amino acid, and/or

any of the codons AGU, AGC, UCG, UCA, UCU coding for Ser may be exchanged by the codon UCC encoding the same amino acid, and/or

any of the codons ACG, ACA, ACU coding for Thr may be exchanged by the codon ACC encoding the same amino acid, and/or

any of the codons GUG, GUA, GUU coding for Val may be exchanged by the codon GUC encoding the same amino acid, and/or

the codon UAU coding for Tyr may be exchanged by the codon UAC encoding the same amino acid.

In any of the above instances, the number of cytosines is increased by 1 per exchanged codon. Exchange of all non C-optimized codons (corresponding to C-optimizable codons) of the coding region results in a C-maximized coding sequence. In the context of the invention at least 70% of the non C-optimized codons are replaced by C-optimized codons of the wild type sequence are replaced by C-optimized codons, preferably at least 80%, more preferably at least 90% within the coding region. It may be preferred that for some amino acids the percentage of C-optimizable codons replaced by C-optimized codons is less than 70%, while for other amino acids the percentage of replaced codons is higher than 70% to meet the overall percentage of C-optimization of at least 70% of all C-optimizable wild type codons of the coding region.

Preferably, in the C-optimized RNAs of the invention, at least 50% of the C-optimizable wild type codons for any given amino acid are replaced by C-optimized codons, e.g. any modified C-enriched RNA preferably contains at least 50% C-optimized codons at C-optimizable wild type codon positions coding for any single of the above mentioned amino acids Ala, Cys, Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Arg, Ser, Thr, Val and Tyr, preferably at least 60%.

In this context codons coding for amino acids, which are not cytosine content-optimizable and which are, however, encoded by at least two codons, may be used without any further selection process. However, the codon of the wild type sequence that codes for a relatively rare tRNA in the cell, e.g. a human cell, may be exchanged for a codon that codes for a relatively frequent tRNA in the cell, whereby both code for the same amino acid. Accordingly, the relatively rare codon GAA coding for Glu may be exchanged by the relative frequent codon GAG coding for the same amino acid, and/or

the relatively rare codon AAA coding for Lys may be exchanged by the relative frequent codon AAG coding for the same amino acid, and/or

the relatively rare codon CAA coding for Gln is exchanged for the relative frequent codon CAG encoding the same amino acid.

In this context, the amino acids Met (AUG) and Trp (UGG), which are encoded by only one codon each, remain unchanged. Stop codons are not cytosine-content optimized, however, the relatively rare stop codons amber, ochre (UAA, UAG) may be exchanged by the relatively frequent stop codon opal (UGA).

The substitutions listed above may obviously be used individually but also in all possible combinations in order to optimize the cytosine-content of the modified RNA compared to the wild type RNA sequence.

Accordingly, the region of the modified RNA coding for the peptide or protein may be changed compared to the coding region of the wild type RNA in such a way that an amino acid encoded by at least two or more codons, of which one comprises one additional cytosine, such a codon may be exchanged by the C-optimized codon comprising one additional cytosine, whereby the amino acid is unaltered compared to the wild type sequence.

Substitutions, additions or eliminations of bases are preferably carried out using a DNA matrix for preparation of the nucleic acid molecule by techniques of the well known site directed mutagenesis or with an oligonucleotide ligation. In such a process, for preparation of the at least one RNA as defined herein a corresponding DNA molecule may be transcribed in vitro. This DNA matrix preferably comprises a suitable promoter, e.g. a T7 or SP6 promoter, for in vitro transcription, which is followed by the desired nucleotide sequence for the at least one RNA to be prepared and a termination signal for in vitro transcription. The DNA molecule, which forms the matrix of the at least one RNA of interest, may be prepared by fermentative proliferation and subsequent isolation as part of a plasmid which can be replicated in bacteria. Plasmids which may be mentioned as suitable for the present invention are e.g. the plasmids pT7Ts (GenBank accession number U26404; Lai et al., Development 1995, 121: 2349 to 2360), pGEM® series, e.g. pGEM®-1 (GenBank accession number X65300; from Promega) and pSP64 (GenBank accession number X65327); cf. also Mezei and Storts, Purification of PCR Products, in: Griffin and Griffin (ed.), PCR Technology: Current Innovation, CRC Press, Boca Raton, Fla., 2001.

Fragments and Variants

In the context of the invention additionally to the here disclosed peptides and proteins, which show a certain degree of identity of sequence, are incorporated. Therefore fragments and variants of the proteins and peptides as defined herein are disclosed herewith in the context of the present invention.

Furthermore fragments and variants of nucleic acids as defined herein are therefore disclosed herewith in the context of the present invention.

Mono-Bi-Multicistronic, Self Cleaving Peptides Etc:

The coding region of the at least one RNA of the inventive composition may occur as a mono-, di-, or even multicistronic RNA, i.e. an RNA sequence which carries the coding sequences of one, two or more proteins or peptides. Such coding sequences of the di-, or even multicistronic RNAs may be separated by at least one internal ribosome entry site (IRES) sequence. Thus, the at least one RNA according to the invention may further comprise one or more internal ribosome entry site (IRES) sequences or IRES-motifs, which may separate several open reading frames, especially if the RNA encodes for two or more peptides or proteins (bi- or multicistronic RNA). For example, the internal ribosome entry site sequence may be derived from EMCV (encephalomyocarditis virus) or from FMDV (Foot and mouth disease virus). Furthermore self-cleaving signal peptides may be used which induce the cleavage of the resulting polypeptide which comprises several proteins or peptides, e.g. a self-cleaving signal peptide sequence derived from F2A peptide from FMDV.

Combinations of Different Coding Sequences

In a preferred embodiment, the inventive composition comprises at least one, two, three, four, five, six, seven, eight, nine, ten or more RNAs, each comprising at least one, two, three, four, five, six, seven, eight, nine, ten or more coding regions encoding at least one or more cytokine as defined above and/or at least one or more chemokine as defined above, and/or at least one or more suicide gene product as defined above, and/or at least one or more immunogenic peptide or protein as defined above, and/or at least one or more apoptosis inducer as defined above, and/or at least one or more angiogenesis inhibitor as defined above, and/or at least one or more heat shock protein as defined above, and/or at least one or more tumor antigen as defined above, and/or at least one or more β-catenin inhibitor as defined above, and/or at least one or more STING pathway activator as defined above, and/or at least one or more checkpoint modulator as defined above, and/or at least one or more innate immune activator, and/or at least one or more antibody as defined above, and/or at least one dominant negative receptor and/or at least one or more decoy receptor, and/or at least one or more inhibitor of myeloid derived suppressor cells (MDSCs), and/or at least one or more IDO pathway inhibitor, and/or at least one or more protein or peptide that bind apoptosis inhibitors as defined above, or variants or fragments thereof.

Untranslated Regions (UTRs)

By a further embodiment the at least one RNA of the inventive composition preferably comprises at least one of the following structural elements: a 5′- and/or 3′-untranslated region element (UTR element), particularly a 5′-UTR element which comprises or consists of a nucleic acid sequence which is derived from the 5′-UTR of a TOP gene or from a fragment, homolog or a variant thereof, or a 5′- and/or 3′-UTR element which may be derivable from a gene that provides a stable mRNA or from a homolog, fragment or variant thereof; a histone stem-loop structure, preferably a histone stem-loop in its 3′ untranslated region; a 5′-CAP structure; a poly-A tail (poly(A) sequence); or a poly(C) sequence.

In a preferred embodiment the at least one RNA comprises at least one 5′- or 3′-UTR element. In this context an UTR element comprises or consists of a nucleic acid sequence which is derived from the 5′- or 3′-UTR of any naturally occurring gene or which is derived from a fragment, a homolog or a variant of the 5′- or 3′-UTR of a gene. Preferably the 5′- or 3′-UTR element used according to the present invention is heterologous to the coding region of the RNA of the inventive composition. Even if 5′- or 3′-UTR elements derived from naturally occurring genes are preferred, also synthetically engineered UTR elements may be used in the context of the present invention.

In a particularly preferred embodiment the at least one RNA comprises at least one 5′-untranslated region element (5′-UTR element) which comprises or consists of a nucleic acid sequence which is derived from the 5′-UTR of a TOP gene or which is derived from a fragment, homolog or variant of the 5′-UTR of a TOP gene.

It is particularly preferred that the 5′-UTR element does not comprise a TOP-motif or a 5′-TOP, as defined above.

In some embodiments, the nucleic acid sequence of the 5′-UTR element which is derived from a 5′-UTR of a TOP gene terminates at its 3′-end with a nucleotide located at position 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 upstream of the start codon (e.g. A(U/T)G) of the gene or mRNA it is derived from. Thus, the 5′-UTR element does not comprise any part of the protein coding region. Thus, preferably, the only protein coding part of mRNA of the inventive composition is provided by the coding region.

The nucleic acid sequence which is derived from the 5′-UTR of a TOP gene is preferably derived from a eukaryotic TOP gene, preferably a plant or animal TOP gene, more preferably a chordate TOP gene, even more preferably a vertebrate TOP gene, most preferably a mammalian TOP gene, such as a human TOP gene.

For example, the 5′-UTR element is preferably selected from 5′-UTR elements comprising or consisting of a nucleic acid sequence which is derived from a nucleic acid sequence selected from the group consisting of SEQ ID Nos. 1-1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of the patent application WO2013/143700, whose disclosure is incorporated herein by reference, from the homologs of SEQ ID Nos. 1-1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of the patent application WO2013/143700, from a variant thereof, or preferably from a corresponding RNA sequence. The term “homologs of SEQ ID Nos. 1-1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of the patent application WO2013/143700” refers to sequences of other species than homo sapiens, which are homologous to the sequences according to SEQ ID Nos. 1-1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of the patent application WO2013/143700.

In a preferred embodiment, the 5′-UTR element comprises or consists of a nucleic acid sequence which is derived from a nucleic acid sequence extending from nucleotide position 5 (i.e. the nucleotide that is located at position 5 in the sequence) to the nucleotide position immediately 5′ to the start codon (located at the 3′ end of the sequences), e.g. the nucleotide position immediately 5′ to the ATG sequence, of a nucleic acid sequence selected from SEQ ID Nos. 1-1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of the patent application WO2013/143700, from the homologs of SEQ ID Nos. 1-1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of the patent application WO2013/143700 from a variant thereof, or a corresponding RNA sequence. It is particularly preferred that the 5′-UTR element is derived from a nucleic acid sequence extending from the nucleotide position immediately 3′ to the 5′-TOP to the nucleotide position immediately 5′ to the start codon (located at the 3′ end of the sequences), e.g. the nucleotide position immediately 5′ to the ATG sequence, of a nucleic acid sequence selected from SEQ ID Nos. 1-1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of the patent application WO2013/143700, from the homologs of SEQ ID Nos. 1-1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of the patent application WO2013/143700, from a variant thereof, or a corresponding RNA sequence.

In a particularly preferred embodiment, the 5′-UTR element comprises or consists of a nucleic acid sequence which is derived from a 5′-UTR of a TOP gene encoding a ribosomal protein or from a variant of a 5′-UTR of a TOP gene encoding a ribosomal protein. For example, the 5′-UTR element comprises or consists of a nucleic acid sequence which is derived from a 5′-UTR of a nucleic acid sequence according to any of SEQ ID NOs: 67, 170, 193, 244, 259, 554, 650, 675, 700, 721, 913, 1016, 1063, 1120, 1138, and 1284-1360 of the patent application WO2013/143700, a corresponding RNA sequence, a homolog thereof, or a variant thereof as described herein, preferably lacking the 5′-TOP motif. As described above, the sequence extending from position 5 to the nucleotide immediately 5′ to the ATG (which is located at the 3′end of the sequences) corresponds to the 5′-UTR of said sequences.

Preferably, the 5′-UTR element comprises or consists of a nucleic acid sequence which is derived from a 5′-UTR of a TOP gene encoding a ribosomal large protein (RPL) or from a homolog or variant of a 5′-UTR of a TOP gene encoding a ribosomal large protein (RPL). For example, the 5′-UTR element comprises or consists of a nucleic acid sequence which is derived from a 5′-UTR of a nucleic acid sequence according to any of SEQ ID NOs: 67, 259, 1284-1318, 1344, 1346, 1348-1354, 1357, 1358, 1421 and 1422 of the patent application WO2013/143700, a corresponding RNA sequence, a homolog thereof, or a variant thereof as described herein, preferably lacking the 5′-TOP motif.

In a particularly preferred embodiment, the 5′-UTR element comprises or consists of a nucleic acid sequence which is derived from the 5′-UTR of a ribosomal protein Large 32 gene, preferably from a vertebrate ribosomal protein Large 32 (L32) gene, more preferably from a mammalian ribosomal protein Large 32 (L32) gene, most preferably from a human ribosomal protein Large 32 (L32) gene, or from a variant of the 5′-UTR of a ribosomal protein Large 32 gene, preferably from a vertebrate ribosomal protein Large 32 (L32) gene, more preferably from a mammalian ribosomal protein Large 32 (L32) gene, most preferably from a human ribosomal protein Large 32 (L32) gene, wherein preferably the 5′-UTR element does not comprise the 5′-TOP of said gene.

A preferred sequence for a 5′-UTR element corresponds to SEQ ID No. 1368 of the patent application WO2013/143700.

Accordingly, in a particularly preferred embodiment, the 5′-UTR element comprises or consists of a nucleic acid sequence which has an identity of at least about 20%, preferably of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence as mentioned above (according to SEQ ID NO. 10.051 (5′-UTR of human ribosomal protein Large 32 lacking the 5′ terminal oligopyrimidine tract: GGCGCTGCCTACGGAGGTGGCAGCCATCTCCTTCTCGGCATC; corresponding to SEQ ID No. 1368 of the patent application WO2013/143700)) or preferably to a corresponding RNA sequence, or wherein the at least one 5′UTR element comprises or consists of a fragment of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID NO. 10.052 or more preferably to a corresponding RNA sequence, wherein, preferably, the fragment is as described above, i.e. being a continuous stretch of nucleotides representing at least 20% etc. of the full-length 5′-UTR.

Preferably, the fragment exhibits a length of at least about 20 nucleotides or more, preferably of at least about 30 nucleotides or more, more preferably of at least about 40 nucleotides or more. Preferably, the fragment is a functional fragment as described herein.

In some embodiments, the mRNA of the inventive composition comprises a 5′-UTR element which comprises or consists of a nucleic acid sequence which is derived from the 5′-UTR of a vertebrate TOP gene, such as a mammalian, e.g. a human TOP gene, selected from RPSA, RPS2, RPS3, RPS3A, RPS4, RPS5, RPS6, RPS7, RPS8, RPS9, RPS10, RPS11, RPS12, RPS13, RPS14, RPS15, RPS15A, RPS16, RPS17, RPS18, RPS19, RPS20, RPS21, RPS23, RPS24, RPS25, RPS26, RPS27, RPS27A, RPS28, RPS29, RPS30, RPL3, RPL4, RPL5, RPL6, RPL7, RPL7A, RPL8, RPL9, RPL10, RPL10A, RPL11, RPL12, RPL13, RPL13A, RPL14, RPL15, RPL17, RPL18, RPL18A, RPL19, RPL21, RPL22, RPL23, RPL23A, RPL24, RPL26, RPL27, RPL27A, RPL28, RPL29, RPL30, RPL31, RPL32, RPL34, RPL35, RPL35A, RPL36, RPL36A, RPL37, RPL37A, RPL38, RPL39, RPL40, RPL41, RPLP0, RPLP1, RPLP2, RPLP3, RPLP0, RPLP1, RPLP2, EEF1A1, EEF1B2, EEF1D, EEF1G, EEF2, EIF3E, EIF3F, EIF3H, EIF2S3, EIF3C, EIF3K, EIF3EIP, EIF4A2, PABPC1, HNRNPA1, TPT1, TUBB1, UBA52, NPM1, ATP5G2, GNB2L1, NME2, UQCRB, or from a homolog or variant thereof, wherein preferably the 5′-UTR element does not comprise a TOP-motif or the 5′-TOP of said genes, and wherein optionally the 5′-UTR element starts at its 5′-end with a nucleotide located at position 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 downstream of the 5′ terminal oligopyrimidine tract (TOP) and wherein further optionally the 5′-UTR element which is derived from a 5′-UTR of a TOP gene terminates at its 3′-end with a nucleotide located at position 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 upstream of the start codon (A(U/T)G) of the gene it is derived from.

In further particularly preferred embodiments, the 5′-UTR element comprises or consists of a nucleic acid sequence which is derived from the 5′-UTR of a ribosomal protein Large 32 gene (RPL32), a ribosomal protein Large 35 gene (RPL35), a ribosomal protein Large 21 gene (RPL21), an ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1, cardiac muscle (ATP5A1) gene, an hydroxysteroid (17-beta) dehydrogenase 4 gene (HSD17B4), an androgen-induced 1 gene (AIG1), cytochrome c oxidase subunit Vlc gene (COX6C), or a N-acylsphingosine amidohydrolase (acid ceramidase) 1 gene (ASAH1) or from a variant thereof, preferably from a vertebrate ribosomal protein Large 32 gene (RPL32), a vertebrate ribosomal protein Large 35 gene (RPL35), a vertebrate ribosomal protein Large 21 gene (RPL21), a vertebrate ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1, cardiac muscle (ATP5A1) gene, a vertebrate hydroxysteroid (17-beta) dehydrogenase 4 gene (HSD17B4), a vertebrate androgen-induced 1 gene (AIG1), a vertebrate cytochrome c oxidase subunit Vlc gene (COX6C), or a vertebrate N-acylsphingosine amidohydrolase (acid ceramidase) 1 gene (ASAH1) or from a variant thereof, more preferably from a mammalian ribosomal protein Large 32 gene (RPL32), a ribosomal protein Large 35 gene (RPL35), a ribosomal protein Large 21 gene (RPL21), a mammalian ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1, cardiac muscle (ATP5A1) gene, a mammalian hydroxysteroid (17-beta) dehydrogenase 4 gene (HSD17B4), a mammalian androgen-induced 1 gene (AIG1), a mammalian cyto-chrome c oxidase subunit Vlc gene (COX6C), or a mammalian N-acylsphingosine ami-dohydrolase (acid ceramidase) 1 gene (ASAH1) or from a variant thereof, most preferably from a human ribosomal protein Large 32 gene (RPL32), a human ribosomal protein Large 35 gene (RPL35), a human ribosomal protein Large 21 gene (RPL21), a human ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1, cardiac muscle (ATP5A1) gene, a human hydroxysteroid (17-beta) dehydrogenase 4 gene (HSD17B4), a human androgen-induced 1 gene (AIG1), a human cytochrome c oxidase subunit Vlc gene (COX6C), or a human N-acylsphingosine amidohydrolase (acid ceramidase) 1 gene (ASAH1) or from a variant thereof, wherein preferably the 5′-UTR element does not comprise the 5′-TOP of said gene.

In this context particularly preferred are 5′-UTR elements comprising a nucleic acid sequence according to SEQ ID Nos. 10.051-10.054.

Accordingly, in a particularly preferred embodiment, the 5′-UTR element comprises or consists of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID No. 1368, or SEQ ID NOs 1412-1420 of the patent application WO2013/143700, or a corresponding RNA sequence, or wherein the at least one 5′-UTR element comprises or consists of a fragment of a nucleic acid sequence which has an identity of at least about 20%, preferably of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID No. 1368, or SEQ ID NOs 1412-1420 of the patent application WO2013/143700, wherein, preferably, the fragment is as described above, i.e. being a continuous stretch of nucleotides representing at least 20% etc. of the full-length 5′-UTR. Preferably, the fragment exhibits a length of at least about 20 nucleotides or more, preferably of at least about 30 nucleotides or more, more preferably of at least about 40 nucleotides or more. Preferably, the fragment is a functional fragment as described herein.

Accordingly, in a particularly preferred embodiment, the 5′-UTR element comprises or consists of a nucleic acid sequence which has an identity of at least about 20%, preferably of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID No. 10.053 (5′-UTR of ATP5A1 lacking the 5′ terminal oligopyrimidine tract: GCGGCTCGGCCATTTTGTCCCAGTCAGTCCGGAGGCTGCGGCTGCAGAAGTACCGCCTGCG-GAGTAACTGCAAAG; corresponding to SEQ ID No. 1414 of the patent application WO2013/143700 (5′-UTR of ATP5A1 lacking the 5′ terminal oligopyrimidine tract) or preferably to a corresponding RNA sequence, or wherein the at least one 5′UTR element comprises or consists of a fragment of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID NO. 26 (of the patent application WO2013/143700) or more preferably to a corresponding RNA sequence, wherein, preferably, the fragment is as described above, i.e. being a continuous stretch of nucleotides representing at least 20% etc. of the full-length 5′-UTR. Preferably, the fragment exhibits a length of at least about 20 nucleotides or more, preferably of at least about 30 nucleotides or more, more preferably of at least about 40 nucleotides or more. Preferably, the fragment is a functional fragment as described herein.

In a further preferred embodiment, the at least one RNA of the inventive composition further comprises at least one 3′-UTR element which comprises or consists of a nucleic acid sequence derived from the 3′-UTR of a chordate gene, preferably a vertebrate gene, more preferably a mammalian gene, most preferably a human gene, or from a variant of the 3′-UTR of a chordate gene, preferably a vertebrate gene, more preferably a mammalian gene, most preferably a human gene.

The term ‘3′-UTR element’ refers to a nucleic acid sequence which comprises or consists of a nucleic acid sequence that is derived from a 3′-UTR or from a variant of a 3′-UTR. A 3′-UTR element in the sense of the present invention may represent the 3′-UTR of an mRNA. Thus, in the sense of the present invention, preferably, a 3′-UTR element may be the 3′-UTR of an mRNA, preferably of an artificial mRNA, or it may be the transcription template for a 3′-UTR of an mRNA. Thus, a 3′-UTR element preferably is a nucleic acid sequence which corresponds to the 3′-UTR of an mRNA, preferably to the 3′-UTR of an artificial mRNA, such as an mRNA obtained by transcription of a genetically engineered vector construct. Preferably, the 3′-UTR element fulfils the function of a 3′-UTR or encodes a sequence which fulfils the function of a 3′-UTR.

Preferably, the inventive mRNA comprises a 3′-UTR element which may be derivable from a gene that relates to an mRNA with an enhanced half-life (that provides a stable mRNA), for example a 3′-UTR element as defined and described below. Preferably, the 3′-UTR element, is a nucleic acid sequence derived from a 3′-UTR of a gene, which preferably encodes a stable mRNA, or from a homolog, a fragment or a variant of said gene

In a particularly preferred embodiment, the 3′-UTR element comprises or consists of a nucleic acid sequence which is derived from a 3′-UTR of a gene selected from the group consisting of an albumin gene, an α-globin gene, a β-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alpha gene, such as a collagen alpha 1(I) gene, or from a variant of a 3′-UTR of a gene selected from the group consisting of an albumin gene, an α-globin gene, a β-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alpha gene, such as a collagen alpha 1(I) gene according to SEQ ID No. 1369-1390 of the patent application WO2013/143700 whose disclosure is incorporated herein by reference. In a particularly preferred embodiment, the 3′-UTR element comprises or consists of a nucleic acid sequence which is derived from a 3′-UTR of an albumin gene, preferably a vertebrate albumin gene, more preferably a mammalian albumin gene, most preferably a human albumin gene, most preferably a human albumin gene according to SEQ ID NO. 10063 (according SEQ ID No: 1369 of the patent application WO2013/143700). The mRNA sequence may comprise or consist of a nucleic acid sequence which is derived from the 3′-UTR of the human albumin gene according to GenBank Accession number NM_000477.5, or from a fragment or variant thereof.

In this context it is particularly preferred that the mRNA of the inventive composition comprises a 3′-UTR element comprising a corresponding RNA sequence derived from the nucleic acids according to SEQ ID No. 1369-1390 of the patent application WO2013/143700 or a fragment, homolog or variant thereof.

Most preferably the 3′-UTR element comprises the nucleic acid sequence derived from a fragment of the human albumin gene (albumin7 3′UTR) according to SEQ ID NO. 10065 (according to SEQ ID No: 1376 of the patent application WO2013/143700).

In this context it is particularly preferred that the 3′-UTR element of the at least one RNA of the inventive composition comprises or consists of a corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO. 10066.

In another particularly preferred embodiment, the 3′-UTR element comprises or consists of a nucleic acid sequence which is derived from a 3′-UTR of an α-globin gene, preferably a vertebrate α- or β-globin gene, more preferably a mammalian α- or β-globin gene, most preferably a human α- or β-globin gene according to SEQ ID NO. 10055 (corresponding to SEQ ID No. 1370 of the patent application WO2013/143700 (3′-UTR of Homo sapiens hemoglobin, alpha 1 (HBA1))), or according to SEQ ID NO. 10057 (corresponding to SEQ ID No. 1371 of the patent application WO2013/143700 (3′-UTR of Homo sapiens hemoglobin, alpha 2 (HBA2))), and/or according to SEQ ID NO. 10059 (corresponding to SEQ ID No. 1372 of the patent application WO2013/143700 (3′-UTR of Homo sapiens hemoglobin, beta (HBB)).

For example, the 3′-UTR element may comprise or consist of the center, α-complex-binding portion of the 3′-UTR of an α-globin gene, according to SEQ ID NO. 10061 (corresponding to SEQ ID No. 1393 of the patent application WO2013/143700).

In this context it is particularly preferred that the 3′-UTR element of the RNA of the inventive composition comprises or consists of a corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO. 10062, according to the above or a homolog, a fragment or variant thereof.

The term ‘a nucleic acid sequence which is derived from the 3′-UTR of a [ . . . ] gene’ preferably refers to a nucleic acid sequence which is based on the 3′-UTR sequence of a [ . . . ] gene or on a part thereof, such as on the 3′-UTR of an albumin gene, an α-globin gene, a β-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, or a collagen alpha gene, such as a collagen alpha 1(I) gene, preferably of an albumin gene or on a part thereof. This term includes sequences corresponding to the entire 3′-UTR sequence, i.e. the full length 3′-UTR sequence of a gene, and sequences corresponding to a fragment of the 3′-UTR sequence of a gene, such as an albumin gene, α-globin gene, β-globin gene, tyrosine hydroxylase gene, lipoxygenase gene, or collagen alpha gene, such as a collagen alpha 1(I) gene, preferably of an albumin gene.

The term ‘a nucleic acid sequence which is derived from a variant of the 3′-UTR of a [ . . . ] gene’ preferably refers to a nucleic acid sequence which is based on a variant of the 3′-UTR sequence of a gene, such as on a variant of the 3′-UTR of an albumin gene, an α-globin gene, a β-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, or a collagen alpha gene, such as a collagen alpha 1(I) gene, or on a part thereof as described above. This term includes sequences corresponding to the entire sequence of the variant of the 3′-UTR of a gene, i.e. the full length variant 3′-UTR sequence of a gene, and sequences corresponding to a fragment of the variant 3′-UTR sequence of a gene. A fragment in this context preferably consists of a continuous stretch of nucleotides corresponding to a continuous stretch of nucleotides in the full-length variant 3′-UTR, which represents at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90% of the full-length variant 3′-UTR. Such a fragment of a variant, in the sense of the present invention, is preferably a functional fragment of a variant as described herein.

Preferably, the at least one 5′-UTR element and the at least one 3′-UTR element act synergistically to increase protein production from the RNA of the inventive composition as described above.

Histone Stem Loop:

In a particularly preferred embodiment, the at least one RNA of the inventive composition comprises a histone stem-loop sequence/structure. Such histone stem-loop sequences are preferably selected from histone stem-loop sequences as disclosed in WO 2012/019780, whose disclosure is incorporated herewith by reference.

A histone stem-loop sequence, suitable to be used within the present invention, is preferably selected from at least one of the following formulae (I) or (II):

formula (I) (stem-loop sequence without stem bordering elements):

formula (II) (stem-loop sequence with stem bordering elements):

wherein:

-   stem1 or stem2 bordering elements N₁₋₆ is a consecutive sequence of     1 to 6, preferably of 2 to 6, more preferably of 2 to 5, even more     preferably of 3 to 5, most preferably of 4 to 5 or 5 N, wherein each     N is independently from another selected from a nucleotide selected     from A, U, T, G and C, or a nucleotide analogue thereof; -   stem1 [N₀₋₂GN₃₋₅] is reverse complementary or partially reverse     complementary with element stem2, and is a consecutive sequence     between of 5 to 7 nucleotides;

wherein N₀₋₂ is a consecutive sequence of 0 to 2, preferably of 0 to 1, more preferably of 1 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C or a nucleotide analogue thereof;

wherein N₃₋₅ is a consecutive sequence of 3 to 5, preferably of 4 to 5, more preferably of 4 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C or a nucleotide analogue thereof, and

wherein G is guanosine or an analogue thereof, and may be optionally replaced by a cytidine or an analogue thereof, provided that its complementary nucleotide cytidine in stem2 is replaced by guanosine;

-   loop sequence [N₀₋₄(U/T)N₀₋₄] is located between elements stem1 and     stem2, and is a consecutive sequence of 3 to 5 nucleotides, more     preferably of 4 nucleotides;

wherein each N₀₋₄ is independent from another a consecutive sequence of 0 to 4, preferably of 1 to 3, more preferably of 1 to 2 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C or a nucleotide analogue thereof; and wherein U/T represents uridine, or optionally thymidine;

-   stem2 [N₃₋₅ CN₀₋₂] is reverse complementary or partially reverse     complementary with element stem1, and is a consecutive sequence     between of 5 to 7 nucleotides;

wherein N₃₋₅ is a consecutive sequence of 3 to 5, preferably of 4 to 5, more preferably of 4 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C or a nucleotide analogue thereof;

wherein N₀₋₂ is a consecutive sequence of 0 to 2, preferably of 0 to 1, more preferably of 1 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G or C or a nucleotide analogue thereof; and

wherein C is cytidine or an analogue thereof, and may be optionally replaced by a guanosine or an analogue thereof provided that its complementary nucleoside guanosine in stem1 is replaced by cytidine;

wherein stem1 and stem2 are capable of base pairing with each other forming a reverse complementary sequence, wherein base pairing may occur between stem1 and stem2, e.g. by Watson-Crick base pairing of nucleotides A and U/T or G and C or by non-Watson-Crick base pairing e.g. wobble base pairing, reverse Watson-Crick base pairing, Hoogsteen base pairing, reverse Hoogsteen base pairing or are capable of base pairing with each other forming a partially reverse complementary sequence, wherein an incomplete base pairing may occur between stem1 and stem2, on the basis that one or more bases in one stem do not have a complementary base in the reverse complementary sequence of the other stem.

According to a further preferred embodiment of the first inventive aspect, the inventive mRNA sequence may comprise at least one histone stem-loop sequence according to at least one of the following specific formulae (Ia) or (IIa):

formula (Ia) (stem-loop sequence without stem bordering elements):

formula (IIa) (stem-loop sequence with stem bordering elements):

wherein N, C, G, T and U are as defined above.

According to a further more particularly preferred embodiment of the first aspect, the at least one RNA may comprise at least one histone stem-loop sequence according to at least one of the following specific formulae (Ib) or (IIb):

formula (Ib) (stem-loop sequence without stem bordering elements):

formula (IIb) (stem-loop sequence with stem bordering elements):

wherein N, C, G, T and U are as defined above.

A particular preferred histone stem-loop sequence is the sequence according to SEQ ID No: 8.

More preferably the stem-loop sequence is the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO: 9

Poly(A)

In a particularly preferred embodiment, the at least one RNA of the inventive composition comprises additionally to the coding region encoding at least one peptide or protein as described above or a fragment or variant thereof, a poly(A) sequence, also called poly-A tail, preferably at the 3′ terminus of the RNA. When present, such a poly(A) sequence comprises a sequence of about 25 to about 400 adenosine nucleotides, preferably a sequence of about 50 to about 400 adenosine nucleotides, more preferably a sequence of about 50 to about 300 adenosine nucleotides, even more preferably a sequence of about 50 to about 250 adenosine nucleotides, most preferably a sequence of about 60 to about 250 adenosine nucleotides. In this context the term “about” refers to a deviation of ±10% of the value(s) it is attached to. This poly(A) sequence is preferably located 3′ of the coding region comprised in the RNA according to the invention.

Preferably, the poly(A) sequence in at least one RNA of the composition is derived from a DNA template by RNA in vitro transcription. Alternatively, the poly(A) sequence may also be obtained in vitro by common methods of chemical-synthesis without being necessarily transcribed from a DNA-progenitor. Moreover, poly(A) sequences, or poly(A) tails may be generated by enzymatic polyadenylation of the at least one RNA using commercially available polyadenylation kits and corresponding protocols known in the art.

Alternatively, the at least one RNA of the inventive composition optionally comprises a polyadenylation signal, which is defined herein as a signal, which conveys polyadenylation to a (transcribed) RNA by specific protein factors (e.g. cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), cleavage factors I and II (CF I and CF II), poly(A) polymerase (PAP)). In this context, a consensus polyadenylation signal is preferred comprising the NN(U/T)ANA consensus sequence. In a particularly preferred aspect, the polyadenylation signal comprises one of the following sequences: AA(U/T)AAA or A(U/T)(U/T)AAA (wherein uridine is usually present in RNA and thymidine is usually present in DNA).

Poly(C)

According to a further preferred embodiment, the RNA of the inventive composition can be modified by a sequence of at least 10 cytosines, preferably at least 20 cytosines, more preferably at least 30 cytosines (so-called “poly(C) sequence”). Particularly, the RNA may contain a poly(C) sequence of typically about 10 to 200 cytosine nucleotides, preferably about 10 to 100 cytosine nucleotides, more preferably about 10 to 70 cytosine nucleotides or even more preferably about 20 to 50 or even 20 to 30 cytosine nucleotides. This poly(C) sequence is preferably located 3′ of the coding region, more preferably 3′ of an optional poly(A) sequence comprised in the RNA according to the present invention.

5′-Cap

According to another preferred embodiment of the invention, a modified RNA molecule as defined herein, can be modified by the addition of a so-called “5′ cap” structure, which preferably stabilizes the RNA as described herein. A 5′-cap is an entity, typically a modified nucleotide entity, which generally “caps” the 5′-end of a mature mRNA. A 5′-cap may typically be formed by a modified nucleotide, particularly by a derivative of a guanine nucleotide. Preferably, the 5′-cap is linked to the 5′-terminus via a 5′-5′-triphosphate linkage. A 5′-cap may be methylated, e.g. m7GpppN, wherein N is the terminal 5′ nucleotide of the nucleic acid carrying the 5′-cap, typically the 5′-end of an mRNA. m7GpppN is the 5′-cap structure, which naturally occurs in mRNA transcribed by polymerase II and is therefore preferably not considered as modification comprised in a modified RNA in this context. Accordingly, a modified RNA of the present invention may comprise a m7GpppN as 5′-cap, but additionally the modified RNA typically comprises at least one further modification as defined herein.

Further examples of 5′cap structures include glyceryl, inverted deoxy abasic residue (moiety), 4′,5′ methylene nucleotide, 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides, alpha-nucleotide, modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3′,4′-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety, 3′-3′-inverted abasic moiety, 3′-2′-inverted nucleotide moiety, 3′-2′-inverted abasic moiety, 1,4-butanediol phosphate, 3′-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3′-phosphate, 3′phosphorothioate, phosphorodithioate, or bridging or non-bridging methylphosphonate moiety. These modified 5′-cap structures are regarded as at least one modification in this context.

Particularly preferred modified 5′-cap structures are cap1 (methylation of the ribose of the adjacent nucleotide of m7G), cap2 (additional methylation of the ribose of the 2nd nucleotide downstream of the m7G), cap3 (additional methylation of the ribose of the 3rd nucleotide downstream of the m7G), cap4 (methylation of the ribose of the 4th nucleotide downstream of the m7G), ARCA (anti-reverse cap analogue, modified ARCA (e.g. phosphothioate modified ARCA), inosine, N1-methyl-guanosine, 2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

Secretory Signal Sequence:

According to another particularly preferred embodiment, the at least one RNA of the composition may additionally or alternatively encode a secretory signal peptide. Such secretory signal sequences are peptide stretches, which typically exhibit a length of about 15 to 30 amino acids and are preferably located at the N-terminus of the encoded peptide, without being limited thereto. Secretory signal sequences as defined herein preferably allow the transport of the encoded peptide or protein as encoded by the at least one coding sequence of the at least one RNA of the composition into a defined cellular compartiment, preferably the cell surface, the endoplasmic reticulum (ER) or the endosomal-lysosomal compartiment. Examples of secretory signal sequences as defined herein include, without being limited thereto, secretory signal sequences of classical or non-classical MHC-molecules (e.g. signal sequences of MHC I and II molecules, e.g. of the MHC class I molecule HLA-A*0201), secretory signal sequences of cytokines or immunoglobulines as defined herein, secretory signal sequences of the invariant chain of immunoglobulines or antibodies as defined herein, signal sequences of Lamp1, Tapasin, Erp57, Calretikulin, Calnexin, and further membrane associated proteins or of proteins associated with the endoplasmic reticulum (ER) or the endosomal-lysosomal compartiment.

Any of the above modifications regarding the coding sequence and/or regarding the RNA as defined above may be applied to the coding sequence and/or the RNA of the composition of the present invention, and further to any RNA as used in the context of the present invention and may be, if suitable or necessary, be combined with each other in any combination, provided, these combinations of modifications do not interfere with each other in the respective at least one RNA. A person skilled in the art will be able to take his choice accordingly.

Production of mRNA and RNA

The RNA may be prepared using any method known in the art, including synthetic methods (chemical synthesis of RNA) such as e.g. solid phase synthesis, as well as in vitro methods, such as RNA in vitro transcription reactions.

Combinations:

According to the present invention it is particularly preferred to combine RNA encoded peptides or proteins. In this context particularly preferred are the following combinations:

-   -   RNA, preferably mRNA coding for at least one cytokine+RNA,         preferably mRNA coding for at least one chemokine     -   RNA, preferably mRNA coding for at least one cytokine+RNA,         preferably mRNA coding for at least one suicide gene product     -   RNA, preferably mRNA coding for at least one cytokine+RNA,         preferably mRNA coding for at least one immunogenic protein or         peptide     -   RNA, preferably mRNA coding for at least one cytokine+RNA,         preferably mRNA coding for at least one apoptosis inducer     -   RNA, preferably mRNA coding for at least one cytokine+RNA,         preferably mRNA coding for at least one angiogenesis inhibitor     -   RNA, preferably mRNA coding for at least one cytokine+RNA,         preferably mRNA coding for at least one heat shock protein     -   RNA, preferably mRNA coding for at least one cytokine+RNA,         preferably mRNA coding for at least one tumor antigen     -   RNA, preferably mRNA coding for at least one cytokine+RNA,         preferably mRNA coding for at least one β-catenin inhibitor     -   RNA, preferably mRNA coding for at least one cytokine+RNA,         preferably mRNA coding for at least one activator of the STING         pathway     -   RNA, preferably mRNA coding for at least one cytokine+RNA,         preferably mRNA coding for at least one checkpoint modulator     -   RNA, preferably mRNA coding for at least one cytokine+RNA,         preferably mRNA coding for at least one innate immune activator     -   RNA, preferably mRNA coding for at least one cytokine+RNA,         preferably mRNA coding for at least one antibody     -   RNA, preferably mRNA coding for at least one cytokine+RNA,         preferably mRNA coding for at least one decoy receptor     -   RNA, preferably mRNA coding for at least one cytokine+RNA,         preferably mRNA coding for at least one inhibitor of myeloid         derived suppressor cells (MDSCs)     -   RNA, preferably mRNA coding for at least one cytokine+RNA,         preferably mRNA coding for at least one IDO pathway inhibitor     -   RNA, preferably mRNA coding for at least one cytokine+RNA,         preferably mRNA coding for at least one protein or peptide that         bind inhibitors of apoptosis.

Furthermore, particularly preferred are the following embodiments:

-   -   RNA, preferably mRNA coding for IL-2 and/or RNA, preferably mRNA         coding for IL-12+mRNA coding for thymidine kinase (approach:         cytokines+suicide gene product)     -   RNA, preferably mRNA coding for IL-2 and/or RNA, preferably mRNA         coding for IL-12     -   RNA, preferably mRNA coding for IL-12 and/or RNA, preferably         mRNA coding for CD40L     -   RNA, preferably mRNA coding for IL-15 and/or RNA, preferably         mRNA coding for IL-12     -   RNA, preferably mRNA coding for IL-2+RNA, preferably mRNA coding         for Influenza NP protein     -   RNA, preferably mRNA coding for IL-2 and/or RNA, preferably mRNA         coding for IL-12+RNA, preferably mRNA coding for cytochrome         c/caspase 3 (cytokines+apoptosis induction)     -   RNA, preferably mRNA coding for CD40L+RNA, preferably mRNA         coding for IL-12+RNA, preferably mRNA coding for ΔRIGI

It has to be understood that the RNA molecules of the inventive composition may code for one or more different peptides or proteins (e.g. cytokines, chemokines, suicide gene products, immunogenic proteins or peptides, apoptosis inducers, angiogenesis inhibitors, heat shock proteins, tumor antigens, β-catenin inhibitors, activators of the STING pathway, checkpoint modulators, innate immune activators, antibodies, dominant negative receptors and decoy receptors, inhibitors of myeloid derived suppressor cells (MDSCs), IDO pathway inhibitors, and proteins or peptides that bind inhibitors of apoptosis. as described above. Several RNA sequences may be combined in one inventive RNA containing composition. Moreover it is possible that the RNA sequence or sequences of the inventive composition code for variants or fragments of the wild type protein sequence or for one or more parts or fragments of the wild type protein sequence or variants thereof.

Non Coding RNA

According to the invention the at least one RNA of the inventive RNA containing composition may comprise at least one non-coding RNA, which is preferably selected from the group consisting of small interfering RNA (siRNA), antisense RNA (asRNA), circular RNA (circRNA), ribozymes, aptamers, riboswitches, immunostimulating/immunostimulatory RNA RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), and Piwi-interacting RNA (piRNA).

Immunostimulatory/Immunostimulating RNA (isRNA):

Likewise, according to a further alternative, the at least one RNA of the inventive RNA containing composition is an immunostimulatory/immunostimulating RNA, which preferably elicits an innate immune response. Such an immunostimulatory RNA may be any (double-stranded or single-stranded) RNA, e.g. a coding RNA, as defined herein. In a preferred embodiment, the immunostimulatory RNA is a non-coding RNA. Preferably, the immunostimulatory RNA may be a single-stranded, a double-stranded or a partially double-stranded RNA, more preferably a single-stranded RNA, and/or a circular or linear RNA, more preferably a linear RNA. More preferably, the immunostimulatory RNA may be a (linear) single-stranded RNA. Even more preferably, the immunostimulatory RNA may be a (long) (linear) single-stranded) non-coding RNA. In this context it is particular preferred that the isRNA carries a triphosphate at its 5′-end which is the case for in vitro transcribed RNA. An immunostimulatory RNA may also occur as a short RNA oligonucleotide as defined herein.

An immunostimulatory RNA as used herein may furthermore be selected from any class of RNA molecules, found in nature or being prepared synthetically, and which can induce an innate immune response and may support an adaptive immune response induced by an antigen. In this context, an immune response may occur in various ways. A substantial factor for a suitable (adaptive) immune response is the stimulation of different T cell sub-populations. T-lymphocytes are typically divided into two sub-populations, the T-helper 1 (Th1) cells and the T-helper 2 (Th2) cells, with which the immune system is capable of destroying intracellular (Th1) and extracellular (Th2) pathogens (e.g. antigens). The two Th cell populations differ in the pattern of the effector proteins (cytokines) produced by them. Thus, Th1 cells assist the cellular immune response by activation of macrophages and cytotoxic T cells. Th2 cells, on the other hand, promote the humoral immune response by stimulation of B-cells for conversion into plasma cells and by formation of antibodies (e.g. against antigens). The Th1/Th2 ratio is therefore of great importance in the induction and maintenance of an adaptive immune response. In connection with the present invention, the Th1/Th2 ratio of the (adaptive) immune response is preferably shifted in the direction towards the cellular response (Th1 response) and a cellular immune response is thereby induced. According to one example, the innate immune system which may support an adaptive immune response may be activated by ligands of Toll-like receptors (TLRs). TLRs are a family of highly conserved pattern recognition receptor (PRR) polypeptides that recognize pathogen-associated molecular patterns (PAMPs) and play a critical role in innate immunity in mammals. Currently at least thirteen family members, designated TLR1-TLR13 (Toll-like receptors: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13), have been identified. Furthermore, a number of specific TLR ligands have been identified. Furthermore, it has been reported that ligands for certain TLRs include certain nucleic acid molecules and that certain types of RNA are immunostimulatory in a sequence-independent or sequence-dependent manner, wherein these various immunostimulatory RNAs may e.g. stimulate TLR3, TLR7, or TLR8, or intracellular receptors such as RIG-I, MDA-5, etc.

Preferably, an immunostimulatory nucleic acid, preferably an immunostimulatory RNA (isRNA), as used herein, may comprise any RNA sequence known to be immunostimulatory, including, without being limited thereto, RNA sequences representing and/or encoding ligands of TLRs, preferably selected from human family members TLR1-TLR10 or murine family members TLR1-TLR13, more preferably selected from (human) family members TLR1-TLR10, even more preferably from TLR7 and TLR8, ligands for intracellular receptors for RNA (such as RIG-I or MDA-5, etc.) (see e.g. Meylan, E., Tschopp, J. (2006). Toll-like receptors and RNA helicases: two parallel ways to trigger antiviral responses. Mol. Cell 22, 561-569), or any other immunostimulatory RNA sequence. Furthermore, (classes of) immunostimulatory RNA molecules, used as a further compound of the inventive vaccine, may include any other RNA capable of eliciting an immune response. Without being limited thereto, such an immunostimulatory RNA may include ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA), and viral RNA (vRNA). Such an immunostimulatory RNA may comprise a length of 1000 to 5000, of 500 to 5000, of 5 to 5000, or of 5 to 1000, 5 to 500, 5 to 250, of 5 to 100, of 5 to 50 or of 5 to 30 nucleotides.

An immunostimulatory RNA as used herein may furthermore be selected from any class of RNA molecules, found in nature or being prepared synthetically, and which can induce an innate immune response and may support an adaptive immune response induced by an antigen. In this context, an immune response may occur in various ways. A substantial factor for a suitable (adaptive) immune response is the stimulation of different T-cell sub-populations. T-lymphocytes are typically divided into two sub-populations, the T-helper 1 (Th1) cells and the T-helper 2 (Th2) cells, with which the immune system is capable of destroying intracellular (Th1) and extracellular (Th2) pathogens (e.g. antigens). The two Th cell populations differ in the pattern of the effector proteins (cytokines) produced by them. Thus, Th1 cells assist the cellular immune response by activation of macrophages and cytotoxic T-cells. Th2 cells, on the other hand, promote the humoral immune response by stimulation of B-cells for conversion into plasma cells and by formation of antibodies (e.g. against antigens). The Th1/Th2 ratio is therefore of great importance in the induction and maintenance of an adaptive immune response. In connection with the present invention, the Th1/Th2 ratio of the (adaptive) immune response is preferably shifted in the direction towards the cellular response (Th1 response) and a cellular immune response is thereby induced. According to one example, the innate immune system which may support an adaptive immune response, may be activated by ligands of Toll-like receptors (TLRs). TLRs are a family of highly conserved pattern recognition receptor (PRR) polypeptides that recognize pathogen-associated molecular patterns (PAMPs) and play a critical role in innate immunity in mammals. Currently at least thirteen family members, designated TLR1-TLR13 (Toll-like receptors: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13), have been identified. Furthermore, a number of specific TLR ligands have been identified. It was e.g. found that unmethylated bacterial DNA and synthetic analogs thereof (CpG DNA) are ligands for TLR9 (Hemmi H et al. (2000) Nature 408:740-5; Bauer S et al. (2001) Proc Natl Acad Sci USA 98, 9237-42). Furthermore, it has been reported that ligands for certain TLRs include certain nucleic acid molecules and that certain types of RNA are immunostimulatory in a sequence-independent or sequence-dependent manner, wherein these various immunostimulatory RNAs may e.g. stimulate TLR3, TLR7, or TLR8, or intracellular receptors such as RIG-I, MDA-5, etc. E.g. Lipford et al. determined certain G,U-containing oligoribonucleotides as immunostimulatory by acting via TLR7 and TLR8 (see WO 03/086280). The immunostimulatory G,U-containing oligoribonucleotides described by Lipford et al. were believed to be derivable from RNA sources including ribosomal RNA, transfer RNA, messenger RNA, and viral RNA.

According to a particularly preferred embodiment, such immunostimulatory nucleic acid sequences is preferably RNA preferably consisting of or comprising a nucleic acid of the following formula (III) or (IV): GIXmGn,  (formula (III))

wherein:

G is guanosine, uracil or an analogue of guanosine or uracil;

X is guanosine, uracil, adenosine, thymidine, cytosine or an analogue of the above-mentioned nucleotides;

I is an integer from 1 to 40,

wherein

when I=1 G is guanosine or an analogue thereof,

when I>1 at least 50% of the nucleotides are guanosine or an analogue thereof;

m is an integer and is at least 3;

wherein

when m=3 X is uracil or an analogue thereof,

-   -   when m>3 at least 3 successive uracils or analogues of uracil         occur;

n is an integer from 1 to 40,

wherein

when n=1 G is guanosine or an analogue thereof,

when n>1 at least 50% of the nucleotides are guanosine or an analogue thereof. CIXmCn,  (formula (IV))

wherein:

C is cytosine, uracil or an analogue of cytosine or uracil;

X is guanosine, uracil, adenosine, thymidine, cytosine or an analogue of the above-mentioned nucleotides;

I is an integer from 1 to 40,

wherein

when I=1 C is cytosine or an analogue thereof,

when I>1 at least 50% of the nucleotides are cytosine or an analogue thereof;

m is an integer and is at least 3;

wherein

when m=3 X is uracil or an analogue thereof,

when m>3 at least 3 successive uracils or analogues of uracil occur;

n is an integer from 1 to 40,

wherein

when n=1 C is cytosine or an analogue thereof,

when n>1 at least 50% of the nucleotides are cytosine or an analogue thereof.

The nucleic acids of formula (II) or (III), which may be used as immunostimulatory RNA may be relatively short nucleic acid molecules with a typical length of approximately from 5 to 100 (but may also be longer than 100 nucleotides for specific embodiments, e.g. up to 200 nucleotides), from 5 to 90 or from 5 to 80 nucleotides, preferably a length of approximately from 5 to 70, more preferably a length of approximately from 8 to 60 and, more preferably a length of approximately from 15 to 60 nucleotides, more preferably from 20 to 60, most preferably from 30 to 60 nucleotides. If the nucleic acid of the inventive nucleic acid cargo complex has a maximum length of e.g. 100 nucleotides, m will typically be <=98. The number of nucleotides G in the nucleic acid of formula (III) is determined by I or n. I and n, independently of one another, are each an integer from 1 to 40, wherein when I or n=1 G is guanosine or an analogue thereof, and when I or n>1 at least 50% of the nucleotides are guanosine or an analogue thereof. For example, without implying any limitation, when I or n=4 GI or Gn can be, for example, a GUGU, GGUU, UGUG, UUGG, GUUG, GGGU, GGUG, GUGG, UGGG or GGGG, etc.; when I or n=5 GI or Gn can be, for example, a GGGUU, GGUGU, GUGGU, UGGGU, UGGUG, UGUGG, UUGGG, GUGUG, GGGGU, GGGUG, GGUGG, GUGGG, UGGGG, or GGGGG, etc.; etc. A nucleotide adjacent to Xm in the nucleic acid of formula (III) according to the invention is preferably not a uracil. Similarly, the number of nucleotides C in the nucleic acid of formula (IV) according to the invention is determined by I or n. I and n, independently of one another, are each an integer from 1 to 40, wherein when I or n=1 C is cytosine or an analogue thereof, and when I or n>1 at least 50% of the nucleotides are cytosine or an analogue thereof. For example, without implying any limitation, when I or n=4, CI or Cn can be, for example, a CUCU, CCUU, UCUC, UUCC, CUUC, CCCU, CCUC, CUCC, UCCC or CCCC, etc.; when I or n=5 CI or Cn can be, for example, a CCCUU, CCUCU, CUCCU, UCCCU, UCCUC, UCUCC, UUCCC, CUCUC, CCCCU, CCCUC, CCUCC, CUCCC, UCCCC, or CCCCC, etc.; etc. A nucleotide adjacent to Xm in the nucleic acid of formula (III) according to the invention is preferably not a uracil. Preferably, for formula (II), when I or n>1, at least 60%, 70%, 80%, 90% or even 100% of the nucleotides are guanosine or an analogue thereof, as defined above. The remaining nucleotides to 100% (when guanosine constitutes less than 100% of the nucleotides) in the flanking sequences G1 and/or Gn are uracil or an analogue thereof, as defined hereinbefore. Also preferably, I and n, independently of one another, are each an integer from 2 to 30, more preferably an integer from 2 to 20 and yet more preferably an integer from 2 to 15. The lower limit of I or n can be varied if necessary and is at least 1, preferably at least 2, more preferably at least 3, 4, 5, 6, 7, 8, 9 or 10. This definition applies correspondingly to formula (III).

According to a particularly preferred embodiment, a nucleic acid according to any of formulas (III) or (IV) above, which may be used as immunostimulatory RNA, may be selected from a sequence consisting or comprising any of the following sequences SEQ ID NOs 298-381.

or from a sequence having at least 60%, 70%, 80%, 90%, or even 95% sequence identity with any of these sequences

According to a further particularly preferred embodiment, such immunostimulatory nucleic acid sequences particularly isRNA consist of or comprise a nucleic acid of formula (V) or (VI): (NuGIXmGnNv)a,  (formula (V))

wherein:

G is guanosine (guanine), uridine (uracil) or an analogue of guanosine (guanine) or uridine (uracil), preferably guanosine (guanine) or an analogue thereof;

X is guanosine (guanine), uridine (uracil), adenosine (adenine), thymidine (thymine), cytidine (cytosine), or an analogue of these nucleotides (nucleosides), preferably uridine (uracil) or an analogue thereof;

N is a nucleic acid sequence having a length of about 4 to 50, preferably of about 4 to 40, more preferably of about 4 to 30 or 4 to 20 nucleic acids, each N independently being selected from guanosine (guanine), uridine (uracil), adenosine (adenine), thymidine (thymine), cytidine (cytosine) or an analogue of these nucleotides (nucleosides);

a is an integer from 1 to 20, preferably from 1 to 15, most preferably from 1 to 10;

I is an integer from 1 to 40,

wherein when I=1, G is guanosine (guanine) or an analogue thereof,

-   -   when I>1, at least 50% of these nucleotides (nucleosides) are         guanosine (guanine) or an analogue thereof;

m is an integer and is at least 3;

wherein when m=3, X is uridine (uracil) or an analogue thereof, and

-   -   when m>3, at least 3 successive uridines (uracils) or analogues         of uridine (uracil) occur;

n is an integer from 1 to 40,

wherein when n=1, G is guanosine (guanine) or an analogue thereof,

-   -   when n>1, at least 50% of these nucleotides (nucleosides) are         guanosine (guanine) or an analogue thereof;

u,v may be independently from each other an integer from 0 to 50,

preferably wherein when u=0, v≥1, or

-   -   when v=0, u 1;

wherein the nucleic acid molecule of formula (IV) has a length of at least 50 nucleotides, preferably of at least 100 nucleotides, more preferably of at least 150 nucleotides, even more preferably of at least 200 nucleotides and most preferably of at least 250 nucleotides. (NuCIXmCnNv)a,  (formula (VI))

wherein:

C is cytidine (cytosine), uridine (uracil) or an analogue of cytidine (cytosine) or uridine (uracil), preferably cytidine (cytosine) or an analogue thereof;

X is guanosine (guanine), uridine (uracil), adenosine (adenine), thymidine (thymine), cytidine (cytosine) or an analogue of the above-mentioned nucleotides (nucleosides), preferably uridine (uracil) or an analogue thereof;

N is each a nucleic acid sequence having independent from each other a length of about 4 to 50, preferably of about 4 to 40, more preferably of about 4 to 30 or 4 to 20 nucleic acids, each N independently being selected from guanosine (guanine), uridine (uracil), adenosine (adenine), thymidine (thymine), cytidine (cytosine) or an analogue of these nucleotides (nucleosides);

a is an integer from 1 to 20, preferably from 1 to 15, most preferably from 1 to 10;

I is an integer from 1 to 40,

wherein when I=1, C is cytidine (cytosine) or an analogue thereof,

-   -   when I>1, at least 50% of these nucleotides (nucleosides) are         cytidine (cytosine) or an analogue thereof;

m is an integer and is at least 3;

wherein when m=3, X is uridine (uracil) or an analogue thereof,

-   -   when m>3, at least 3 successive uridines (uracils) or analogues         of uridine (uracil) occur;

n is an integer from 1 to 40,

wherein when n=1, C is cytidine (cytosine) or an analogue thereof,

-   -   when n>1, at least 50% of these nucleotides (nucleosides) are         cytidine (cytosine) or an analogue thereof.

u, v may be independently from each other an integer from 0 to 50,

preferably wherein when u=0, v≥1, or

-   -   when v=0, u≥1;

wherein the nucleic acid molecule of formula (V) according to the invention has a length of at least 50 nucleotides, preferably of at least 100 nucleotides, more preferably of at least 150 nucleotides, even more preferably of at least 200 nucleotides and most preferably of at least 250 nucleotides.

For formula (VI), any of the definitions given above for elements N (i.e. Nu and Nv) and X (Xm), particularly the core structure as defined above, as well as for integers a, I, m, n, u and v, similarly apply to elements of formula (VI) correspondingly, wherein in formula (VI) the core structure is defined by CIXmCn. The definition of bordering elements Nu and Nv is identical to the definitions given above for Nu and Nv.

According to a very particularly preferred embodiment, the nucleic acid molecule, preferably immunostimulating RNA according to formula (V) may be selected from e.g. any of the sequences according to SEQ ID NOs 382-395 or from a sequence having at least 60%, 70%, 80%, 90%, or even 95% sequence identity with any of these sequences.

In this context particularly preferred are immunostimulating RNAs according to SEQ ID NOs 5, 394 and 10072.

R2025:

(SEQ ID NO: 5 and 394) GGGAGAAAGCUCAAGCUUAUCCAAGUAGGCUGGUCACCUGUACAACGUAG CCGGUAUUUUUUUUUUUUUUUUUUUUUUGACCGUCUCAAGGUCCAAGUUA GUCUGCCUAUAAAGGUGCGGAUCCACAGCUGAUGAAAGACUUGUGCGGUA CGGUUAAUCUCCCCUUUUUUUUUUUUUUUUUUUUUAGUAAAUGCGUCUAC UGAAUCCAGCGAUGAUGCUGGCCCAGAUCUUCGACCACAAGUGCAUAUAG UAGUCAUCGAGGGUCGCCUUUUUUUUUUUUUUUUUUUUUUUGGCCCAGUU CUGAGACUUCGCUAGAGACUACAGUUACAGCUGCAGUAGUAACCACUGCG GCUAUUGCAGGAAAUCCCGUUCAGGUUUUUUUUUUUUUUUUUUUUUCCGC UCACUAUGAUUAAGAACCAGGUGGAGUGUCACUGCUCUCGAGGUCUCACG AGAGCGCUCGAUACAGUCCUUGGAAGAAUCUUUUUUUUUUUUUUUUUUUU UUGUGCGACGAUCACAGAGAACUUCUAUUCAUGCAGGUCUGCUCUAG 

R3630:

(SEQ ID NO. 10072) GGGAGAAAGCUCAAGCUUAUCCAAGUAGGCUGGUCACCUGUACAACGUAG CCGGUAUUUUUUUUUUUUUUUUUUUUUUGACCGUCUCAAGGUCCAAGUUA GUCUGCCUAUAAAGGUGCGGAUCCACAGCUGAUGAAAGACUUGUGCGGUA CGGUUAAUCUCCCCUUUUUUUUUUUUUUUUUUUUUAGUAAAUGCGUCUAC UGAAUCCAGCGAUGAUGCUGGCCCAGAUCUUCGACCACAAGUGCAUAUAG UAGUCAUCGAGGGUCGCCUUUUUUUUUUUUUUUUUUUUUUUGGCCCAGUU CUGAGACUUCGCUAGAGACUACAGUUACAGCUGCAGUAGUAACCACUGCG GCUAUUGCAGGAAAUCCCGUUCAGGUUUUUUUUUUUUUUUUUUUUUCCGC UCACUAUGAUUAAGAACCAGGUGGAGUGUCACUGCUCUCGAGGUCUCACG AGAGCGCUCGAUACAGUGCUUGGAAGAAUCUUUUUUUUUUUUUUUUUUUU UUGUGCGACGAUCACAGAGAACUUCUAUUCAUGCAGGUCUGCUCUAG.

According to another very particularly preferred embodiment, the nucleic acid molecule according to formula (VI) may be selected from e.g. any of the sequences according to SEQ ID NO 396 or 397, or from a sequence having at least 60%, 70%, 80%, 90%, or even 95% sequence identity with any of these sequences.

All modifications disclosed in the context of coding RNA may also be applied in the context of non-coding RNA if applicable.

Combination of Coding and Non-Coding RNA

In particularly preferred embodiments the inventive RNA containing composition comprises at least one RNA encoding at least one peptide or protein and at least one non-coding RNA as defined above, preferably at least one immunostimulating RNA.

Particularly preferred are the following embodiments:

-   -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,         or 10072+RNA, preferably mRNA coding for at least one cytokine,         preferably IL-2, IL-12, IL-15 or CD40L     -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,         or 10072+RNA, preferably mRNA coding for at least one chemokine     -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,         or 10072+RNA, preferably mRNA coding for at least one suicide         gene product, preferably Herpes simplex virus thymidine kinase     -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,         or 10072+RNA, preferably mRNA coding for at least one         immunogenic protein or peptide, preferably Influenza NP protein     -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,         or 10072+RNA, preferably mRNA coding for at least one apoptosis         inducer, preferably cytochrome c or caspase 3     -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,         or 10072+RNA, preferably mRNA coding for at least one         angiogenesis inducer     -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,         or 10072+RNA, preferably mRNA coding for at least one heat shock         protein     -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,         or 10072+RNA, preferably mRNA coding for at least one tumor         antigen     -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,         or 10072+RNA, preferably mRNA coding for at least one β-catenin         inhibitor     -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,         or 10072+RNA, preferably mRNA coding for at least one activator         of the STING pathway     -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,         or 10072+RNA, preferably mRNA coding for at least one checkpoint         modulator, preferably an antibody directed against PD-1, PD-L1         or CTLA4     -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,         or 10072+RNA, preferably mRNA coding for at least one innate         immune activator, preferably a constitutive active variant of         RIG-1     -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,         or 10072+RNA, preferably mRNA coding for at least one antibody     -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,         or 10072+RNA, preferably mRNA coding for at least one decoy         receptor, preferably a soluble PD-1 receptor     -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,         or 10072+RNA, preferably mRNA coding for at least one inhibitor         of myeloid derived suppressor cells (MDSCs)     -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,         or 10072+RNA, preferably mRNA coding for at least one IDO         pathway inhibitor     -   Immostimulating RNA preferably according to SEQ ID Nos XY or         YY+RNA, preferably mRNA coding for at least one protein or         peptide that bind inhibitors of apoptosis.

More particularly preferred are the following embodiments:

-   -   Immostimulating RNA preferably according SEQ ID Nos 5, 394, or         10072+RNA, preferably mRNA coding for at least one cytokine,         preferably IL-2, IL-12, IL-15 or CD40L+RNA, preferably mRNA         coding for at least one immunogenic protein or peptide,         preferably Influenza NP protein     -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,         or 10072+RNA, preferably mRNA coding for at least one cytokine,         preferably IL-2, IL-12, IL-15 or CD40L+RNA, preferably mRNA         coding for at least one innate immune activator, preferably a         constitutive active variant of RIG-1     -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,         or 10072+RNA, preferably mRNA coding for at least one cytokine,         preferably IL-2, IL-12, or IL-15, +RNA, preferably mRNA coding         for at least one further cytokine, preferably CD40L.

Formulation and Complexation

The at least one RNA of the inventive composition may be administered naked without being associated with any further vehicle, carrier, transfection or complexation agent.

In a preferred embodiment, the RNA of the inventive composition is formulated together with further compounds for increasing the transfection efficiency and/or the immunostimulatory properties of the RNA. Such compounds are termed herein carriers, vehicles, transfection or complexation agents. Preferably, the RNA is formulated together with one or more cationic or polycationic compounds, preferably with cationic or polycationic polymers, cationic or polycationic peptides or proteins, cationic or polycationic polysaccharides, cationic or polycationic lipids and/or with a polymeric carrier. Such cationic or polycationic polymers, cationic or polycationic peptides or proteins, cationic or polycationic polysaccharides, cationic or polycationic lipids or polymeric carriers are useful as carriers, vehicles, transfection or complexation agents of nucleic acids in the context of the present invention. Accordingly, in a further embodiment of the invention it is preferred that the at least one RNA or any other nucleic acid comprised in the inventive composition is associated with or complexed with a cationic or polycationic compound or a polymeric carrier, optionally in a weight ratio selected from a range of about 6:1 (w/w) to about 0.25:1 (w/w), more preferably from about 5:1 (w/w) to about 0.5:1 (w/w), even more preferably of about 4:1 (w/w) to about 1:1 (w/w) or of about 3:1 (w/w) to about 1:1 (w/w), and most preferably a ratio of about 3:1 (w/w) to about 2:1 (w/w) of RNA or nucleic acid to cationic or polycationic compound and/or with a polymeric carrier; or optionally in a nitrogen/phosphate ratio of RNA or nucleic acid to cationic or polycationic compound and/or polymeric carrier in the range of about 0.1-10, preferably in a range of about 0.3-4 or 0.3-1, and most preferably in a range of about 0.5-1 or 0.7-1, and even most preferably in a range of about 0.3-0.9 or 0.5-0.9.

The ratio of the at least one RNA as described above, and the cationic or polycationic compound, may be calculated on the basis of the nitrogen/phosphate ratio (N/P-ratio) of all these components. In the context of the present invention, an N/P-ratio is preferably in the range of about 0.01-4, 0.01-2, 0.1-2 or 0.1-1.5 regarding the ratio of nucleic acids:cationic or polycationic peptide contained in the inventive vaccine, and most preferably in the range of about 0.1-1. Such an N/P ratio is preferably designed to provide good transfection properties in vivo and transport into and through cell membranes. Preferably, for this purpose, cationic or polycationic compound and/or polymeric carriers as used herein, are based on peptide sequences.

Cationic or polycationic compounds, being particularly preferred agents in this context include protamine, nucleoline, spermine or spermidine, or other cationic peptides or proteins, such as poly-L-lysine (PLL), poly-arginine, basic polypeptides, cell penetrating peptides (CPPs), including HIV-binding peptides, HIV-1 Tat (HIV), Tat-derived peptides, Penetratin, VP22 derived or analog peptides, HSV VP22 (Herpes simplex), MAP, KALA or protein transduction domains (PTDs), PpT620, proline-rich peptides, arginine-rich peptides, lysine-rich peptides, MPG-peptide(s), Pep-1, L-oligomers, Calcitonin peptide(s), Antennapedia-derived peptides (particularly from Drosophila antennapedia), pAntp, pIsI, FGF, Lactoferrin, Transportan, Buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, or histones.

In this context protamine is particularly preferred.

Additionally, preferred cationic or polycationic proteins or peptides may be selected from the following proteins or peptides having the following total formula (VII): (Arg)_(I);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x),  (formula (VII)

wherein I+m+n+o+x=8-15, and I, m, n or o independently of each other may be any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, provided that the overall content of Arg, Lys, His and Orn represents at least 50% of all amino acids of the oligopeptide; and Xaa may be any amino acid selected from native (=naturally occurring) or non-native amino acids except of Arg, Lys, His or Orn; and x may be any number selected from 0, 1, 2, 3 or 4, provided, that the overall content of Xaa does not exceed 50% of all amino acids of the oligopeptide. Particularly preferred cationic peptides in this context are e.g. Arg₇, Arg₈, Arg₉, H₃R₉, R₉H₃, H₃R₉H₃, YSSR₉SSY, (RKH)₄, Y(RKH)₂R, etc. In this context the disclosure of WO 2009/030481 is incorporated herewith by reference.

A polymeric carrier used according to the invention might be a polymeric carrier formed by disulfide-crosslinked cationic components.

According to a further particularly preferred embodiment, cationic or polycationic peptides or proteins of the polymeric carrier, having the empirical sum formula (VII) as shown above and which comprise or are additionally modified to comprise at least one —SH moeity, may be, without being restricted thereto, selected from the subgroup consisting of generic formulas Arg7 (also termed as R7), Arg9 (also termed R9), Arg12 (also termed as R12).

According to a one further particularly preferred embodiment, the cationic or polycationic peptide or protein of the polymeric carrier, when defined according to formula {(Arg)I;(Lys)m;(His)n;(Orn)o;(Xaa)x} (formula (VII)) as shown above and which comprise or are additionally modified to comprise at least one —SH moeity, may be, without being restricted thereto, selected from subformula (VIIa): {(Arg)I;(Lys)m;(His)n;(Orn)o;(Xaa′)x(Cys)y}  formula (VIIa)

wherein (Arg)I;(Lys)m;(His)n;(Orn)o; and x are as defined herein, Xaa′ is any amino acid selected from native (=naturally occurring) or non-native amino acids except of Arg, Lys, His, Orn or Cys and y is any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80 and 81-90, provided that the overall content of Arg (Arginine), Lys (Lysine), His (Histidine) and Orn (Ornithine) represents at least 10% of all amino acids of the oligopeptide.

This embodiment may apply to situations, wherein the cationic or polycationic peptide or protein of the polymeric carrier, e.g. when defined according to empirical formula (Arg)I;(Lys)m;(His)n;(Orn)o;(Xaa)x (formula (VII)) as shown above, comprises or has been modified with at least one cysteine as —SH moiety in the above meaning such that the cationic or polycationic peptide as cationic component carries at least one cysteine, which is capable to form a disulfide bond with other components of the polymeric carrier.

Exemplary examples may comprise any of the following sequences:

Cys(Arg7), Cys(Arg8), Cys(Arg9), Cys(Arg10), Cys(Arg11), Cys(Arg12), Cys(Arg13), Cys(Arg14), Cys(Arg15), Cys(Arg16), Cys(Arg17), Cys(Arg18), Cys(Arg19), Cys(Arg20).

According to another particularly preferred embodiment, the cationic or polycationic peptide or protein of the polymeric carrier, when defined according to formula {(Arg)I;(Lys)m;(His)n;(Orn)o;(Xaa)x} (formula (VII)) as shown above, may be, without being restricted thereto, selected from subformula (VIIb): Cys1{(Arg)I;(Lys)m;(His)n;(Orn)o;(Xaa)x}Cys2  (formula (VIIb))

wherein empirical formula {(Arg)I;(Lys)m;(His)n;(Orn)o;(Xaa)x} (formula (VII)) is as defined herein and forms a core of an amino acid sequence according to (semiempirical) formula (I) and wherein Cys1 and Cys2 are Cysteines proximal to, or terminal to (Arg)I;(Lys)m;(His)n;(Orn)o;(Xaa)x. Exemplary examples may comprise any of the above sequences flanked by two Cys and following sequences:

Cys(Arg7)Cys, Cys(Arg8)Cys, Cys(Arg9)Cys, Cys(Arg10)Cys, Cys(Arg11)Cys, Cys(Arg12)Cys, Cys(Arg13)Cys, Cys(Arg14)Cys, Cys(Arg15)Cys, Cys(Arg16)Cys, Cys(Arg17)Cys, Cys(Arg18)Cys, Cys(Arg19)Cys, Cys(Arg20)Cys (SEQ ID NOs: 10-23):

This embodiment may apply to situations, wherein the cationic or polycationic peptide or protein of the polymeric carrier, e.g. when defined according to empirical formula (Arg)I;(Lys)m;(His)n;(Orn)o;(Xaa)x (formula (VII)) as shown above, has been modified with at least two cysteines as —SH moieties in the above meaning such that the cationic or polycationic peptide of the inventive polymeric carrier cargo complex as cationic component carries at least two (terminal) cysteines, which are capable to form a disulfide bond with other components of the polymeric carrier.

In a preferred embodiment, the polymeric carrier is formed by, comprises or consists of the peptide CysArg12Cys (SEQ ID NO: 15) or CysArg12 (CRRRRRRRRRRRR).

According to a second alternative, at least one cationic (or polycationic) component of the polymeric carrier may be selected from e.g. any (non-peptidic) cationic or polycationic polymer suitable in this context, provided that this (non-peptidic) cationic or polycationic polymer exhibits or is modified to exhibit at least one —SH-moiety, which provide for a disulfide bond linking the cationic or polycationic polymer with another component of the polymeric carrier as defined herein. Thus, likewise as defined herein, the polymeric carrier may comprise the same or different cationic or polycationic polymers.

In the specific case that the cationic component of the polymeric carrier comprises a (non-peptidic) cationic or polycationic polymer the cationic properties of the (non-peptidic) cationic or polycationic polymer may be determined upon its content of cationic charges when compared to the overall charges of the components of the cationic polymer. Preferably, the content of cationic charges in the cationic polymer at a (physiological) pH as defined herein is at least 10%, 20%, or 30%, preferably at least 40%, more preferably at least 50%, 60% or 70%, but also preferably at least 80%, 90%, or even 95%, 96%, 97%, 98%, 99% or 100%, most preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, or may be in the range of about 10% to 90%, more preferably in the range of about 30% to 100%, even preferably in the range of about 50% to 100%, e.g. 50, 60, 70, 80%, 90% or 100%, or in a range formed by any two of the afore mentioned values, provided, that the content of all charges, e.g. positive and negative charges at a (physiological) pH as defined herein, in the entire cationic polymer is 100%.

Preferably, the (non-peptidic) cationic component of the polymeric carrier represents a cationic or polycationic polymer, typically exhibiting a molecular weight of about 0.1 or 0.5 kDa to about 100 kDa, preferably of about 1 kDa to about 75 kDa, more preferably of about 5 kDa to about 50 kDa, even more preferably of about 5 kDa to about 30 kDa, or a molecular weight of about 10 kDa to about 50 kDa, even more preferably of about 10 kDa to about 30 kDa. Additionally, the (non-peptidic) cationic or polycationic polymer typically exhibits at least one —SH-moiety, which is capable to form a disulfide linkage upon condensation with either other cationic components or other components of the polymeric carrier as defined herein.

In the above context, the (non-peptidic) cationic component of the polymeric carrier may be selected from acrylates, modified acrylates, such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), chitosanes, aziridines or 2-ethyl-2-oxazoline (forming oligo ethylenimines or modified oligoethylenimines), polymers obtained by reaction of bisacrylates with amines forming oligo beta aminoesters or poly amido amines, or other polymers like polyesters, polycarbonates, etc. Each molecule of these (non-peptidic) cationic or polycationic polymers typically exhibits at least one —SH-moiety, wherein these at least one —SH-moiety may be introduced into the (non-peptidic) cationic or polycationic polymer by chemical modifications, e.g. using imonothiolan, 3-thio propionic acid or introduction of —SH-moieties containing amino acids, such as cysteine or any further (modified) amino acid. Such —SH-moieties are preferably as already defined above.

The disulfide-crosslinked cationic components may be the same or different from each other. The polymeric carrier can also contain further components. It is also particularly preferred that the polymeric carrier used according to the present invention comprises mixtures of cationic peptides, proteins or polymers and optionally further components as defined herein, which are crosslinked by disulfide bonds as described herein. In this context the disclosure of WO 2012/013326 is incorporated herewith by reference.

In this context the cationic components, which form basis for the polymeric carrier by disulfide-crosslinkage, are typically selected from any suitable cationic or polycationic peptide, protein or polymer suitable for this purpose, particular any cationic or polycationic peptide, protein or polymer capable to complex an RNA or a nucleic acid as defined according to the present invention, and thereby preferably condensing the RNA or the nucleic acid. The cationic or polycationic peptide, protein or polymer, is preferably a linear molecule, however, branched cationic or polycationic peptides, proteins or polymers may also be used.

Every disulfide-crosslinking cationic or polycationic protein, peptide or polymer of the polymeric carrier, which may be used to complex the RNA of the inventive composition or any further nucleic acid comprised in the inventive composition contains at least one —SH moiety, most preferably at least one cysteine residue or any further chemical group exhibiting an —SH moiety, capable to form a disulfide linkage upon condensation with at least one further cationic or polycationic protein, peptide or polymer as cationic component of the polymeric carrier as mentioned herein.

As defined above, the polymeric carrier, which may be used to complex the RNA of the inventive composition or any further nucleic acid comprised in the inventive composition may be formed by disulfide-crosslinked cationic (or polycationic) components.

Nucleic acids complexed with such polymeric carriers are also termed herein as “polymeric carrier cargo complexes”.

In this context it is particularly preferred that if immunostimulating RNA is used in the context of the present invention that this immunostimulating RNA is complexed with a polymeric carrier as defined above. Preferably, the immunostimulating RNA, (e.g. comprising an RNA sequence according to any of formulae III-VI), most preferably comprising an RNA sequence according to SEQ ID NOs. 5, 394, or 10072, is complexed with a polymeric carrier comprising or formed by disulfide-crosslinked peptides according to formula VII, VIIa or VIIb, preferably a polymeric carrier formed by Cys(Arg12)Cys or Cys(Arg12). Such a particularly preferred embodiment is termed herein also as “RNAdjuvant”.

In a further particular embodiment, the polymeric carrier which may be used to complex the RNA or any further nucleic acid comprised in the inventive composition may be selected from a polymeric carrier molecule according to generic formula (VIII): L-P¹—S—[S—P²—S]_(n)—S—P³-L  formula (VIII)

wherein,

-   P¹ and P³ are different or identical to each other and represent a     linear or branched hydrophilic polymer chain, each P¹ and P³     exhibiting at least one —SH-moiety, capable to form a disulfide     linkage upon condensation with component P², or alternatively with     (AA), (AA)_(x), or [(AA)_(x)]_(z) if such components are used as a     linker between P¹ and P² or P³ and P² and/or with further components     (e.g. (AA), (AA)_(x), [(AA)_(x)]_(z), or L), the linear or branched     hydrophilic polymer chain selected independent from each other from     polyethylene glycol (PEG), poly-N-(2-hydroxypropyl)methacrylamide,     poly-2-(methacryloyloxy)ethyl phosphorylcholines, poly(hydroxyalkyl     L-asparagine), poly(2-(methacryloyloxy)ethyl phosphorylcholine),     hydroxyethylstarch or poly(hydroxyalkyl L-glutamine), wherein the     hydrophilic polymer chain exhibits a molecular weight of about 1 kDa     to about 100 kDa, preferably of about 2 kDa to about 25 kDa; or more     preferably of about 2 kDa to about 10 kDa, e.g. about 5 kDa to about     25 kDa or 5 kDa to about 10 kDa; -   P² is a cationic or polycationic peptide or protein, e.g. as defined     above for the polymeric carrier formed by disulfide-crosslinked     cationic components, and preferably having a length of about 3 to     about 100 amino acids, more preferably having a length of about 3 to     about 50 amino acids, even more preferably having a length of about     3 to about 25 amino acids, e.g. a length of about 3 to 10, 5 to 15,     10 to 20 or 15 to 25 amino acids, more preferably a length of about     5 to about 20 and even more preferably a length of about 10 to about     20; or     -   is a cationic or polycationic polymer, e.g. as defined above for         the polymeric carrier formed by disulfide-crosslinked cationic         components, typically having a molecular weight of about 0.5 kDa         to about 30 kDa, including a molecular weight of about 1 kDa to         about 20 kDa, even more preferably of about 1.5 kDa to about 10         kDa, or having a molecular weight of about 0.5 kDa to about 100         kDa, including a molecular weight of about 10 kDa to about 50         kDa, even more preferably of about 10 kDa to about 30 kDa;     -   each P² exhibiting at least two —SH-moieties, capable to form a         disulfide linkage upon condensation with further components P²         or component(s) P¹ and/or P³ or alternatively with further         components (e.g. (AA), (AA)_(x), or [(AA)_(x)]_(z)); -   —S—S— is a (reversible) disulfide bond (the brackets are omitted for     better readability), wherein S preferably represents sulphur or a     —SH carrying moiety, which has formed a (reversible) disulfide bond.     The (reversible) disulfide bond is preferably formed by condensation     of —SH-moieties of either components P¹ and P², P² and P², or P² and     P³, or optionally of further components as defined herein (e.g. L,     (AA), (AA)_(x), [(AA)_(x)]_(z), etc); The —SH-moiety may be part of     the structure of these components or added by a modification as     defined below; -   L is an optional ligand, which may be present or not, and may be     selected independent from the other from RGD, Transferrin, Folate, a     signal peptide or signal sequence, a localization signal or     sequence, a nuclear localization signal or sequence (NLS), an     antibody, a cell penetrating peptide, (e.g. TAT or KALA), a ligand     of a receptor (e.g. cytokines, hormones, growth factors etc), small     molecules (e.g. carbohydrates like mannose or galactose or synthetic     ligands), small molecule agonists, inhibitors or antagonists of     receptors (e.g. RGD peptidomimetic analogues), or any further     protein as defined herein, etc.; -   n is an integer, typically selected from a range of about 1 to 50,     preferably from a range of about 1, 2 or 3 to 30, more preferably     from a range of about 1, 2, 3, 4, or 5 to 25, or a range of about 1,     2, 3, 4, or 5 to 20, or a range of about 1, 2, 3, 4, or 5 to 15, or     a range of about 1, 2, 3, 4, or 5 to 10, including e.g. a range of     about 4 to 9, 4 to 10, 3 to 20, 4 to 20, 5 to 20, or 10 to 20, or a     range of about 3 to 15, 4 to 15, 5 to 15, or 10 to 15, or a range of     about 6 to 11 or 7 to 10. Most preferably, n is in a range of about     1, 2, 3, 4, or 5 to 10, more preferably in a range of about 1, 2, 3,     or 4 to 9, in a range of about 1, 2, 3, or 4 to 8, or in a range of     about 1, 2, or 3 to 7.

In this context the disclosure of WO 2011/026641 and WO 2012/116811 is incorporated herewith by reference. Each of hydrophilic polymers P¹ and P³ typically exhibits at least one —SH-moiety, wherein the at least one —SH-moiety is capable to form a disulfide linkage upon reaction with component P² or with component (AA) or (AA)_(x), if used as linker between P¹ and P² or P³ and P² as defined below and optionally with a further component, e.g. L and/or (AA) or (AA)_(x), e.g. if two or more —SH-moieties are contained. The following subformulae “P¹—S—S—P²” and “P²—S—S—P³” within the generic formula above, wherein any of S, P¹ and P³ are as defined herein, typically represent a situation, wherein one —SH-moiety of hydrophilic polymers P¹ and P³ was condensed with one —SH-moiety of component P² of the generic formula above, wherein both sulphurs of these —SH-moieties form a disulfide bond —S—S—. These —SH-moieties are typically provided by each of the hydrophilic polymers P¹ and P³, e.g. via an internal cysteine or any further (modified) amino acid or compound which carries a —SH moiety. Accordingly, the subformulae “P¹—S—S—P²” and “P²—S—S—P³” may also be written as “P¹-Cys-Cys-P²” and “P²-Cys-Cys-P³”, if the —SH-moiety is provided by a cysteine, wherein the term Cys-Cys represents two cysteines coupled via a disulfide bond, not via a peptide bond. In this case, the term “—S—S—” in these formulae may also be written as “—S-Cys”, as “-Cys-S” or as “-Cys-Cys-”. In this context, the term “-Cys-Cys-” does not represent a peptide bond but a linkage of two cysteines via their —SH-moieties to form a disulfide bond. Accordingly, the term “-Cys-Cys-” also may be understood generally as “-(Cys-S)—(S-Cys)-”, wherein in this specific case S indicates the sulphur of the —SH-moiety of cysteine. Likewise, the terms “—S-Cys” and “-Cys-S” indicate a disulfide bond between a —SH containing moiety and a cysteine, which may also be written as “—S—(S-Cys)” and “-(Cys-S)—S”. Alternatively, the hydrophilic polymers P¹ and P³ may be modified with a —SH moiety, preferably via a chemical reaction with a compound carrying a —SH moiety, such that each of the hydrophilic polymers P¹ and P³ carries at least one such —SH moiety. Such a compound carrying a —SH moiety may be e.g. an (additional) cysteine or any further (modified) amino acid, which carries a —SH moiety. Such a compound may also be any non-amino compound or moiety, which contains or allows to introduce a —SH moiety into hydrophilic polymers P¹ and P³ as defined herein. Such non-amino compounds may be attached to the hydrophilic polymers P¹ and P³ of the polymeric carrier via chemical reactions or binding of compounds, e.g. by binding of a 3-thio propionic acid or thioimolane, by amide formation (e.g. carboxylic acids, sulphonic acids, amines, etc), by Michael addition (e.g maleinimide moieties, unsatured carbonyls, etc), by click chemistry (e.g. azides or alkines), by alkene/alkine methatesis (e.g. alkenes or alkines), imine or hydrozone formation (aldehydes or ketons, hydrazins, hydroxylamins, amines), complexation reactions (avidin, biotin, protein G) or components which allow S_(n)-type substitution reactions (e.g halogenalkans, thiols, alcohols, amines, hydrazines, hydrazides, sulphonic acid esters, oxyphosphonium salts) or other chemical moieties which can be utilized in the attachment of further components. A particularly preferred PEG derivate in this context is alpha-Methoxy-omega-mercapto poly(ethylene glycol). In each case, the SH-moiety, e.g. of a cysteine or of any further (modified) amino acid or compound, may be present at the terminal ends or internally at any position of hydrophilic polymers P¹ and P³. As defined herein, each of hydrophilic polymers P¹ and P³ typically exhibits at least one —SH-moiety preferably at one terminal end, but may also contain two or even more —SH-moieties, which may be used to additionally attach further components as defined herein, preferably further functional peptides or proteins e.g. a ligand, an amino acid component (AA) or (AA)_(x), antibodies, cell penetrating peptides or enhancer peptides (e.g. TAT, KALA), etc.

As defined above, ligands (L), may be optionally used in the polymeric carrier molecule according to generic formula (VIII), e.g. for direction of the inventive carrier polymer and its entire “cargo” (the adjuvant component and/or the antigen of the inventive composition or vaccine composition) into specific cells. They may be selected independent from the other from RGD, Transferrin, Folate, a signal peptide or signal sequence, a localization signal or sequence, a nuclear localization signal or sequence (NLS), an antibody, a cell penetrating peptide (CPP), (e.g. TAT, KALA), a ligand of a receptor (e.g. cytokines, hormones, growth factors etc), small molecules (e.g. carbohydrates like mannose or galactose or synthetic ligands), small molecule agonists, inhibitors or antagonists of receptors (e.g. RGD peptidomimetic analogues) or any such molecule as further defined below, etc. Particularly preferred are cell penetrating peptides (CPPs), which induce a pH-mediated conformational change in the endosome and lead to an improved release of the inventive polymeric carrier (in complex with a nucleic acid) from the endosome by insertion into the lipid layer of the liposome. Such called CPPs or cationic peptides for transportation, may include, without being limited thereto protamine, nucleoline, spermine or spermidine, poly-L-lysine (PLL), basic polypeptides, poly-arginine, chimeric CPPs, such as Transportan, or MPG peptides, HIV-binding peptides, Tat, HIV-1 Tat (HIV), Tat-derived peptides, oligoarginines, members of the penetratin family, e.g. Penetratin, Antennapedia-derived peptides (particularly from Drosophila antennapedia), pAntp, pIsI, etc., antimicrobial-derived CPPs e.g. Buforin-2, Bac715-24, Syn B, SynB(1), pVEC, hCT-derived peptides, SAP, MAP, PpTG20, Proline-rich peptides, Loligomers, Arginine-rich peptides, Calcitonin-peptides, FGF, Lactoferrin, poly-L-Lysine, poly-Arginine, histones, VP22 derived or analog peptides, Pestivirus Erns, HSV, VP22 (Herpes simplex), MAP, KALA or protein transduction domains (PTDs, PpT620, prolin-rich peptides, arginine-rich peptides, lysine-rich peptides, Pep-1, L-oligomers, Calcitonin peptide(s), etc. Particularly preferred in this context is mannose as ligand to target antigen presenting cells which carries on their cell membrane mannose receptors. In a further preferred aspect of the first embodiment of the present invention galactose as optional ligand can be used to target hepatocytes. Such ligands may be attached to component P¹ and/or P³ by reversible disulfide bonds as defined below or by any other possible chemical attachment, e.g. by amide formation (e.g. carboxylic acids, sulphonic acids, amines, etc), by Michael addition (e.g. maleinimide moieties, α,β unsatured carbonyls, etc), by click chemistry (e.g. azides or alkines), by alkene/alkine methatesis (e.g. alkenes or alkines), imine or hydrozone formation (aldehydes or ketons, hydrazins, hydroxylamins, amines), complexation reactions (avidin, biotin, protein G) or components which allow S_(n)-type substitution reactions (e.g halogenalkans, thiols, alcohols, amines, hydrazines, hydrazides, sulphonic acid esters, oxyphosphonium salts) or other chemical moieties which can be utilized in the attachment of further components.

In the context of formula (VIII) of the present invention components P¹ and P³ represent a linear or branched hydrophilic polymer chain, containing at least one —SH-moiety, each P¹ and P³ independently selected from each other, e.g. from polyethylene glycol (PEG), poly-N-(2-hydroxypropyl)methacrylamide, poly-2-(methacryloyloxy)ethyl phosphorylcholines, poly(hydroxyalkyl L-asparagine) or poly(hydroxyalkyl L-glutamine). P¹ and P³ may be identical or different to each other. Preferably, each of hydrophilic polymers P¹ and P³ exhibits a molecular weight of about 1 kDa to about 100 kDa, preferably of about 1 kDa to about 75 kDa, more preferably of about 5 kDa to about 50 kDa, even more preferably of about 5 kDa to about 25 kDa. Additionally, each of hydrophilic polymers P¹ and P³ typically exhibits at least one —SH-moiety, wherein the at least one —SH-moiety is capable to form a disulfide linkage upon reaction with component P² or with component (AA) or (AA)_(x), if used as linker between P¹ and P² or P³ and P² as defined below and optionally with a further component, e.g. L and/or (AA) or (AA)_(x), e.g. if two or more —SH-moieties are contained. The following subformulae “P¹—S—S—P²” and “P²—S—S—P³” within generic formula (VII) above (the brackets are omitted for better readability), wherein any of S, P¹ and P³ are as defined herein, typically represent a situation, wherein one —SH-moiety of hydrophilic polymers P¹ and P³ was condensed with one —SH-moiety of component P² of generic formula (VII) above, wherein both sulphurs of these —SH-moieties form a disulfide bond —S—S— as defined herein in formula (VII). These —SH-moieties are typically provided by each of the hydrophilic polymers P¹ and P³, e.g. via an internal cysteine or any further (modified) amino acid or compound which carries a —SH moiety. Accordingly, the subformulae “P¹—S—S—P²” and “P²—S—S—P³” may also be written as “P¹-Cys-Cys-P²” and “P²-Cys-Cys-P³”, if the —SH-moiety is provided by a cysteine, wherein the term Cys-Cys represents two cysteines coupled via a disulfide bond, not via a peptide bond. In this case, the term “—S—S—” in these formulae may also be written as “—S-Cys”, as “-Cys-S” or as “-Cys-Cys-”. In this context, the term “-Cys-Cys-” does not represent a peptide bond but a linkage of two cysteines via their —SH-moieties to form a disulfide bond. Accordingly, the term “-Cys-Cys-” also may be understood generally as “-(Cys-S)—(S-Cys)-”, wherein in this specific case S indicates the sulphur of the —SH-moiety of cysteine. Likewise, the terms “—S-Cys” and “-Cys-S” indicate a disulfide bond between a —SH containing moiety and a cysteine, which may also be written as “—S—(S-Cys)” and “-(Cys-S)—S”. Alternatively, the hydrophilic polymers P¹ and P³ may be modified with a —SH moiety, preferably via a chemical reaction with a compound carrying a —SH moiety, such that each of the hydrophilic polymers P¹ and P³ carries at least one such —SH moiety. Such a compound carrying a —SH moiety may be e.g. an (additional) cysteine or any further (modified) amino acid, which carries a —SH moiety. Such a compound may also be any non-amino compound or moiety, which contains or allows to introduce a —SH moiety into hydrophilic polymers P¹ and P³ as defined herein. Such non-amino compounds may be attached to the hydrophilic polymers P¹ and P³ of formula (VII) of the polymeric carrier according to the present invention via chemical reactions or binding of compounds, e.g. by binding of a 3-thio propionic acid or thioimolane, by amide formation (e.g. carboxylic acids, sulphonic acids, amines, etc), by Michael addition (e.g maleinimide moieties, α,β unsatured carbonyls, etc), by click chemistry (e.g. azides or alkines), by alkene/alkine methatesis (e.g. alkenes or alkines), imine or hydrozone formation (aldehydes or ketons, hydrazins, hydroxylamins, amines), complexation reactions (avidin, biotin, protein G) or components which allow S_(n)-type substitution reactions (e.g halogenalkans, thiols, alcohols, amines, hydrazines, hydrazides, sulphonic acid esters, oxyphosphonium salts) or other chemical moieties which can be utilized in the attachment of further components. A particularly preferred PEG derivate in this context is alpha-Methoxy-omega-mercapto poly(ethylene glycol). In each case, the SH-moiety, e.g. of a cysteine or of any further (modified) amino acid or compound, may be present at the terminal ends or internally at any position of hydrophilic polymers P¹ and P³. As defined herein, each of hydrophilic polymers P¹ and P³ typically exhibits at least one —SH-moiety preferably at one terminal end, but may also contain two or even more —SH-moieties, which may be used to additionally attach further components as defined herein, preferably further functional peptides or proteins e.g. a ligand, an amino acid component (AA) or (AA)_(x), antibodies, cell penetrating peptides or enhancer peptides (e.g. TAT, KALA), etc.

According to one preferred alternative, such further functional peptides or proteins may comprise so called cell penetrating peptides (CPPs) or cationic peptides for transportation. Particularly preferred are CPPs, which induce a pH-mediated conformational change in the endosome and lead to an improved release of the inventive polymeric carrier (in complex with a nucleic acid) from the endosome by insertion into the lipid layer of the liposome. Such called cell penetrating peptides (CPPs) or cationic peptides for transportation, may include, without being limited thereto protamine, nucleoline, spermine or spermidine, poly-L-lysine (PLL), basic polypeptides, poly-arginine, chimeric CPPs, such as Transportan, or MPG peptides, HIV-binding peptides, Tat, HIV-1 Tat (HIV), Tat-derived peptides, oligoarginines, members of the penetratin family, e.g. Penetratin, Antennapedia-derived peptides (particularly from Drosophila antennapedia), pAntp, pIsI, etc., antimicrobial-derived CPPs e.g. Buforin-2, Bac715-24, Syn B, SynB(1), pVEC, hCT-derived peptides, SAP, MAP, PpTG20, Proline-rich peptides, Loligomers, Arginine-rich peptides, Calcitonin-peptides, FGF, Lactoferrin, poly-L-Lysine, poly-Arginine, histones, VP22 derived or analog peptides, Pestivirus Erns, HSV, VP22 (Herpes simplex), MAP, KALA or protein transduction domains (PTDs, PpT620, prolin-rich peptides, arginine-rich peptides, lysine-rich peptides, Pep-1, L-oligomers, Calcitonin peptide(s), etc.

According to a further preferred embodiment of the present invention, each of hydrophilic polymers P¹ and P³ of formula (VIII) of the polymeric carrier used according to the present invention may also contain at least one further functional moiety, which allows attaching further components as defined herein, e.g. a ligand as defined above, or functionalities which allow the attachment of further components, e.g. by amide formation (e.g. carboxylic acids, sulphonic acids, amines, etc), by Michael addition (e.g maleinimide moieties, unsatured carbonyls, etc), by click chemistry (e.g. azides or alkines), by alkene/alkine methatesis (e.g. alkenes or alkines), imine or hydrozone formation (aldehydes or ketons, hydrazins, hydroxylamins, amines), complexation reactions (avidin, biotin, protein G) or components which allow S_(n)-type substitution reactions (e.g halogenalkans, thiols, alcohols, amines, hydrazines, hydrazides, sulphonic acid esters, oxyphosphonium salts) or other chemical moieties which can be utilized in the attachment of further components. Further functional moieties may comprise an amino acid component (AA) as defined herein or (AA)_(x), wherein (AA) is preferably an amino component as defined above. In the above context, x is preferably an integer and may be selected from a range of about 1 to 100, preferably from a range of about 1 to 50, more preferably 1 to 30, and even more preferably selected from a number comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15-30, e.g. from a range of about 1 to 30, from a range of about 1 to 15, or from a number comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, or may be selected from a range formed by any two of the afore mentioned values. Most preferably, x is 1. Such an amino acid component (AA) or (AA)_(x) may be contained in every part of the inventive polymeric carrier according to formula (VIII) above and therefore may be attached to all components of the inventive polymeric carrier according to formula (VII). It is particularly preferred that amino acid component (AA) or (AA)_(x) is present as a ligand or part of the repetitive component [S—P²—S]_(n) within formula (VIII) of the inventive polymeric carrier.

In the context of the entire formula (VIII) of the polymeric carrier may be preferably defined as follows: L-P¹—S-[Cys-P²-Cys]_(n)-S—P³-L wherein L, P¹, P², P³ and n are as defined herein, S is sulphur and each Cys provides for one —SH-moiety for the disulfide bond.

According to a particular embodiment, the polymeric carrier according to formula (VII) as defined above, may comprise at least one amino acid component (AA) or (AA)_(x), as defined above. Such an amino acid component (AA) or (AA)_(x) may be contained in every part of the inventive polymeric carrier according to formula (VIII) above and therefore may be attached to all components of the polymeric carrier according to formula (VIII). It is particularly preferred that amino acid component (AA) or (AA)_(x) is present as a ligand or part of the repetitive component [S—P²—S]_(n) within formula (VIII) of the polymeric carrier. The amino acid component (AA) or (AA)_(x) preferably contains or is flanked (e.g. terminally) by at least one —SH containing moiety, which allows introducing this component (AA) or (AA)_(x) via a disulfide bond into the polymeric carrier according to formula (VIII) as defined herein. Such a —SH-containing moiety may be any —SH containing moiety (or, of course, one sulphur of a disulfide bond), e.g. a cysteine residue. In the specific case that the —SH containing moiety represents a cysteine, the amino acid component (AA)_(x) may also be read as -Cys-(AA)_(x)- or -Cys-(AA)_(x)-Cys- wherein Cys represents Cysteine and provides for the necessary —SH-moiety for a disulfide bond. The —SH containing moiety may be also introduced into the amino acid component (AA)_(x) using any of modifications or reactions as shown above for components P¹, P² or P³. In the specific case that the amino acid component (AA)_(x) is linked to two components of the polymeric carrier according to formula (VIII) it is preferred that (AA) or (AA)_(x) contains at least two —SH-moieties, e.g. at least two Cysteines, preferably at its terminal ends. This is particularly preferred if (AA) or (AA)_(x) is part of the repetitive component [S—P²—S]_(n). Alternatively, the amino acid component (AA) or (AA)_(x) is introduced into the polymeric carrier according to formula (VIII) as defined herein via any chemical possible addition reaction. Therefore the amino acid component (AA) or (AA)_(x) contains at least one further functional moiety, which allows attaching same to a further component as defined herein, e.g. component P¹ or P³, P², L, or a further amino acid component (AA) or (AA)_(x), etc. Such functional moieties may be selected from functionalities which allow the attachment of further components, e.g. functionalities as defined herein, e.g. by amide formation (e.g. carboxylic acids, sulphonic acids, amines, etc), by Michael addition (e.g maleinimide moieties, α,β unsatured carbonyls, etc), by click chemistry (e.g. azides or alkines), by alkene/alkine methatesis (e.g. alkenes or alkines), imine or hydrozone formation (aldehydes or ketons, hydrazins, hydroxylamins, amines), complexation reactions (avidin, biotin, protein G) or components which allow S_(n)-type substitution reactions (e.g halogenalkans, thiols, alcohols, amines, hydrazines, hydrazides, sulphonic acid esters, oxyphosphonium salts) or other chemical moieties which can be utilized in the attachment of further components.

The amino acid component (AA) or (AA)_(x) in the polymeric carrier of formula (VIII) may also occur as a mixed repetitive amino acid component [(AA)_(x)]_(z), wherein the number of amino acid components (AA) or (AA)_(x) is further defined by integer z. In this context, z may be selected from a range of about 1 to 30, preferably from a range of about 1 to 15, more preferably 1 to 10 or 1 to 5 and even more preferably selected from a number selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, or may be selected from a range formed by any two of the afore mentioned values.

According to a specific and particularly preferred alternative, the amino acid component (AA) or (AA)_(x), preferably written as S-(AA)_(x)-S or [S-(AA)_(x)-S] may be used to modify component P², particularly the content of component S—P²—S in repetitive component [S—P²—S]_(n) of the polymeric carrier of formula (VIII) above. This may be represented in the context of the entire polymeric carrier according to formula (VIII) e.g. by following formula (Villa): L-P¹—S—{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}—S—P³-L,

wherein x, S, L, AA, P¹, P² and P³ are preferably as defined herein. In formula (Villa) above, any of the single components [S—P²—S] and [S-(AA)_(x)-S] may occur in any order in the subformula {[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}. The numbers of single components [S—P²—S] and [S-(AA)_(x)-S] in the subformula {[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)} are determined by integers a and b, wherein a+b=n. n is an integer and is defined as above for formula (VIII).

a is an integer, typically selected independent from integer b from a range of about 1 to 50, preferably from a range of about 1, 2 or 3 to 30, more preferably from a range of about 1, 2, 3, 4, or 5 to 25, or a range of about 1, 2, 3, 4, or 5 to 20, or a range of about 1, 2, 3, 4, or 5 to 15, or a range of about 1, 2, 3, 4, or 5 to 10, including e.g. a range of about 3 to 20, 4 to 20, 5 to 20, or 10 to 20, or a range of about 3 to 15, 4 to 15, 5 to 15, or 10 to 15, or a range of about 6 to 11 or 7 to 10. Most preferably, a is in a range of about 1, 2, 3, 4, or 5 to 10, more preferably in a range of about 1, 2, 3, or 4 to 9, in a range of about 1, 2, 3, or 4 to 8, or in a range of about 1, 2, or 3 to 7.

b is an integer, typically selected independent from integer a from a range of about 0 to 50 or 1 to 50, preferably from a range of about 0, 1, 2 or 3 to 30, more preferably from a range of about 0, 1, 2, 3, 4, or 5 to 25, or a range of about 0, 1, 2, 3, 4, or 5 to 20, or a range of about 0, 1, 2, 3, 4, or 5 to 15, or a range of about 0, 1, 2, 3, 4, or 5 to 10, including e.g. a range of about 3 to 20, 4 to 20, 5 to 20, or 10 to 20, or a range of about 3 to 15, 4 to 15, 5 to 15, or 10 to 15, or a range of about 6 to 11 or 7 to 10. Most preferably, b is in a range of about 1, 2, 3, 4, or 5 to 10, more preferably in a range of about 1, 2, 3, or 4 to 9, in a range of about 1, 2, 3, or 4 to 8, or in a range of about 1, 2, or 3 to 7.

In this context it is particularly preferred that the RNA, preferably mRNA of the inventive composition is complexed at least partially with a cationic or polycationic compound and/or a polymeric carrier, preferably cationic proteins or peptides. In this context the disclosure of WO 2010/037539 and WO 2012/113513 is incorporated herewith by reference. Partially means that only a part of the RNA is complexed with a cationic compound and that the rest of the RNA is (comprised in the inventive composition) in uncomplexed form (“free”). Preferably the ratio of complexed RNA to:free RNA (in the inventive composition) is selected from a range of about 5:1 (w/w) to about 1:10 (w/w), more preferably from a range of about 4:1 (w/w) to about 1:8 (w/w), even more preferably from a range of about 3:1 (w/w) to about 1:5 (w/w) or 1:3 (w/w), and most preferably the ratio of complexed RNA to free RNA in the inventive composition is selected from a ratio of about 1:1 (w/w).

The so called “(adjuvant) component”, which may be used to together with the RNA, preferably mRNA in the inventive composition, is preferably prepared according to a first step by complexing the at least one (m)RNA of the (adjuvant) component with a cationic or polycationic compound and/or with a polymeric carrier, preferably as defined herein, in a specific ratio to form a stable complex. In this context, it is highly preferable, that no free cationic or polycationic compound or polymeric carrier or only a neglectably small amount thereof remains in the (adjuvant) component after complexing the (m)RNA. Accordingly, the ratio of the (m)RNA and the cationic or polycationic compound and/or the polymeric carrier in the (adjuvant) component is typically selected in a range that the (m)RNA is entirely complexed and no free cationic or polycationic compound or polymeric carrier or only a neglectably small amount thereof remains in the composition. Preferably the ratio of the (adjuvant) component, i.e. the ratio of the (m)RNA to the cationic or polycationic compound and/or the polymeric carrier, preferably as defined herein, is selected from a range of about 6:1 (w/w) to about 0.25:1 (w/w), more preferably from about 5:1 (w/w) to about 0.5:1 (w/w), even more preferably of about 4:1 (w/w) to about 1:1 (w/w) or of about 3:1 (w/w) to about 1:1 (w/w), and most preferably a ratio of about 3:1 (w/w) to about 2:1 (w/w). Alternatively, the ratio of the (m)RNA to the cationic or polycationic compound and/or the polymeric carrier, preferably as defined herein, in the (adjuvant) component, may also be calculated on the basis of the nitrogen/phosphate ratio (N/P-ratio) of the entire complex. In the context of the present invention, an N/P-ratio is preferably in the range of about 0.1-10, preferably in a range of about 0.3-4 and most preferably in a range of about 0.5-2 or 0.7-2 regarding the ratio of RNA:cationic or polycationic compound and/or polymeric carrier, preferably as defined herein, in the complex, and most preferably in the range of about 0.7-1.5, preferably provided the cationic or polycationic compound in the complex is a cationic or polycationic cationic or polycationic protein or peptide and/or the polymeric carrier is as defined herein. Such ratios, particularly weight and/or N/P ratios may also be applied to ratios of the at least one RNA as defined herein to a cationic or polycationic polymer or a polymeric carrier as defined herein used to complex the at least one RNA.

In this context, the N/P ratio is a measure of the ionic charge of the cationic (side chain) component of the cationic or polycationic compound or. In particular, if the cationic properties of the cationic compound are generated by nitrogens (e.g. of the amino acid side chains), the N/P ratio expresses the ratio of basic nitrogen atoms to phosphate residues in the nucleotide backbone, considering that (side chain) nitrogen atoms in the cationic compound contribute to positive charges and phosphate of the phosphate backbone of the nucleic acid contribute to the negative charge. The N/P-ratio is defined as the nitrogen/phosphate ratio (N/P-ratio) of the entire complex of nucleic acid and cationic or polycationic compound. This is typically illustrative for the content/amount of cationic compounds and characteristic for the content/amount of nucleic acids bound or complexed. It may be calculated on the basis that, for example, 1 μg RNA typically contains about 3 nmol phosphate residues, provided that RNA exhibits a statistical distribution of bases. Additionally, 1 nmol peptide typically contains about x nmol nitrogen residues, dependent on the molecular weight and the number of its (cationic) amino acids.

According to a particularly preferred embodiment the inventive composition comprises a polymeric carrier cargo complex comprising or consisting of

-   -   a) as a carrier a polymeric carrier formed by         disulfide-crosslinked cationic components preferably as defined         above, more preferably according to formula VII, VIIa, VIIb or         VIII, and     -   b) as a cargo at least one nucleic acid molecule, preferably an         immunostimulating RNA, most preferably an RNA comprising an RNA         sequence according to SEQ ID NOs. 5, 394, or 10072, preferably         for use as a medicament, more preferably for use as an         immunostimulating agent or adjuvant, preferably for the         treatment of cancer or tumor diseases, wherein the polymeric         carrier cargo complex is preferably administered intratumorally.

In a preferred embodiment, the inventive RNA containing composition comprises a polymeric carrier cargo complex, comprising:

-   -   a) as a carrier a polymeric carrier formed by         disulfide-crosslinked cationic components, preferably as defined         above, more preferably according to formula VII, VIIa, VIIb or         VIII, and     -   a) b) as a cargo at least one first nucleic acid molecule,         preferably an immunostimulating RNA, most preferably an RNA         comprising an RNA sequence according to SEQ ID NOs. 5, 394, or         10072,

for use as an immunostimulating agent or as an adjuvant,

and at least one second nucleic acid molecule, preferably an RNA and more preferably an mRNA encoding at least one protein or a peptide most preferably as disclosed above for coding RNA, and wherein the inventive composition is preferably administered intratumorally.

In a preferred embodiment, the invention relates to a polymeric carrier cargo complex, comprising:

-   -   a) as a carrier a polymeric carrier formed by         disulfide-crosslinked cationic components, preferably as defined         above, more preferably according to formula VII, VIIa, VIIb or         VIII, and     -   a) b) as a cargo at least one first nucleic acid molecule,         preferably an immunostimulating RNA, most preferably an RNA         comprising an RNA sequence according to SEQ ID NOs. 5, 394, or         10072,

for use as an immunostimulating agent or as an adjuvant,

wherein the polymeric carrier cargo complex is administered in combination with at least one second nucleic acid molecule, preferably an RNA and more preferably an mRNA encoding at least one protein or a peptide most preferably as disclosed above for coding RNA, and

wherein the polymeric carrier cargo complex and the second nucleic acid molecule are preferably administered intratumorally.

Such preferred combinations of at least one first nucleic acid, preferably an immunostimulating RNA and at least one second nucleic acid, preferably an RNA, and more preferably an mRNA encoding at least one protein or peptide are disclosed above in the context of “combinations of coding and non-coding RNA”.

As used herein, the term “first nucleic acid molecule” refers to a nucleic molecule, which is used as a cargo in the polymeric carrier cargo complex and is thus associated with the polymeric carrier. The term “second nucleic acid molecule”, as used herein, typically refers to a nucleic acid, which is not part of the polymeric carrier cargo complex and which encodes at least one peptide or protein.

In the context of the present invention immunostimulating agents or adjuvants are understood as compounds, which are preferably efficient in inducing an innate immune response, particularly in inducing the anti-viral cytokine IFN-alpha.

Adjuvants or immunostimulating agents usually act via their capability to induce an innate immune response. The innate immune system forms the dominant system of host defense in most organisms and comprises barriers such as humoral and chemical barriers including, e.g., inflammation, the complement system and cellular barriers. The innate immune system is typically based on a small number of receptors, called pattern recognition receptors. They recognize conserved molecular patterns that distinguish foreign organisms, like viruses, bacteria, fungi and parasites, from cells of the host. Such pathogen-associated molecular patterns (PAMP) include viral nucleic acids, components of bacterial and fungal walls, flagellar proteins, and more. The first family of pattern recognition receptors (PAMP receptors) studied in detail was the Toll-like receptor (TLR) family. TLRs are transmembrane proteins which recognize ligands of the extracellular milieu or of the lumen of endosomes. Following ligand-binding they transduce the signal via cytoplasmic adaptor proteins which leads to triggering of a host-defence response and entailing production of antimicrobial peptides, proinflammatory chemokines and cytokines, antiviral cytokines, etc. (see e.g. Meylan, E., J. Tschopp, et al. (2006), Nature 442(7098): 39-44). Further relevant components of the immune system include e.g. the endosomal TLRs, cytoplasmic receptors, Type I interferons and cytoplasmic receptors. Therefore, the immunostimulating agents or adjuvants are defined herein preferably as inducers of an innate immune response, which activate pattern recognition receptors (PAMP receptors). Hereby, a cascade of signals is elicited, which e.g. may result in the release of cytokines (e.g. IFN-alpha) supporting the innate immune response. Accordingly, it is preferably a feature of an immunostimulating agent or adjuvant to bind to such receptors and activate such PAMP receptors. Ideally, such as an agent or adjuvant additionally supports the adaptive immune response by e.g. shifting the immune response such that the preferred class of Th cells is activated. Depending on the disease or disorder to be treated a shift to a Th1-based immune response may be preferred or, in other cases, a shift to a Th2 immune response may be preferred. Furthermore, adjuvants are usually defined as compounds that can increase and/or modulate the intrinsic immunogenicity of an antigen.

The term “immunostimulating agent” is typically understood not to include agents as e.g. antigens (of whatever chemical structure), which elicit an adaptive/cytotoxic immune response, e.g. a “humoral” or “cellular” immune response, in other words elicit immune responses (and confer immunity by themselves) which are characterized by a specific response to structural properties of an antigen recognized to be foreign by immune competent cells. Rather “immunostimulating agent” is typically understood to mean agents/compounds/complexes which do not trigger any adaptive immune response by themselves, but which may exclusively enhance such an adaptive immune response in an unspecific way, by e.g. activating “PAMP” receptors and thereby triggering the release of cytokines which support the actual adaptive immune response. Accordingly, any immunostimulation by agents (e.g. antigens) which evoke an adaptive immune response by themselves (conferring immunity by themselves directly or indirectly) is typically disclaimed by the phrase “immunostimulating agent”.

The term “adjuvant” is also understood not to comprise agents which confer immunity by themselves.

Accordingly, adjuvants do not by themselves confer immunity, but assist the immune system in various ways to enhance the antigen-specific immune response by e.g. promoting presentation of an antigen to the immune system. Hereby, an adjuvant may preferably e.g. modulate the antigen-specific immune response by e.g. shifting the dominating Th2-based antigen specific response to a more Th1-based antigen specific response or vice versa. Accordingly, the terms “immunostimulating agent” and “adjuvant” in the context of the present invention are typically understood to mean agents, compounds or complexes which do not confer immunity by themselves, but exclusively support the immune response in an unspecific way (in contrast to an antigen-specific immune response) by effects, which modulate the antigen-specific (adaptive cellular and/or humoral immune response) by unspecific measures, e.g. cytokine expression/secretion, improved antigen presentation, shifting the nature of the arms of the immune response etc. Accordingly, any agents evoking by themselves immunity are typically disclaimed by the terms “adjuvant” or “immunostimulating agent”.

The use of the polymeric carrier cargo complex optionally in combination with a second nucleic acid molecule, preferably an RNA, allows provision of a more efficient and/or safer medicament. Advantageously, the polymeric carrier cargo complex is suited for in vivo delivery of nucleic acids, in particular for compacting and stabilizing a nucleic acid for the purposes of nucleic acid transfection, such as exhibiting one or more reduced negative side effects of high-molecular weight polymers as discussed above, such as poor biodegradability or high toxicity, agglomeration, low transfection activity in vivo, etc. The polymeric carrier cargo complex also provides for improved nucleic acid transfer in vivo, particularly via intratumoral routes, including serum stability, salt stability, efficiency of uptake, reduced complement activation, nucleic acid release, etc. Such a polymeric carrier cargo complex furthermore may support induction and maintenance of an adaptive immune response by initiating or boosting a parallel innate immune response. It has been found that an improved adaptive immune response can further be obtained, in particular when the polymeric carrier cargo complex is administered in combination with a second nucleic acid molecule, preferably an RNA, encoding a protein or peptide, or when the polymeric carrier cargo complex is co-formulated in a pharmaceutical composition with a second nucleic acid molecule, preferably an RNA, encoding a protein or peptide, preferably an antigenic peptide or protein. It has proven as particularly beneficial in this respect to administer the inventive composition comprising the polymeric carrier cargo complex optionally in combination with the second nucleic acid molecule as defined herein via an intratumoral route. Additionally, the polymeric carrier cargo complex may exhibit improved storage stability, particularly during lyophilisation.

In particular embodiments, the polymeric carrier cargo complex as defined above enhances the immune response against a protein or peptide, which is encoded by a second nucleic acid molecule, preferably an RNA, more preferably an mRNA, that is administered in combination with the polymeric carrier cargo complex, preferably via an intratumoral route of administration.

The polymeric carrier cargo complex and/or the second nucleic acid molecule encoding a peptide or protein are preferably provided together with a pharmaceutically acceptable carrier and/or vehicle. In the context of the present invention, a pharmaceutically acceptable carrier typically includes the liquid or non-liquid material, which is mixed with the polymeric carrier cargo complex and/or the second nucleic acid molecule. If the polymeric carrier cargo complex and/or the second nucleic acid molecule are provided in liquid form, the carrier will typically be pyrogen-free water; isotonic saline or buffered aqueous solutions, e.g phosphate, citrate etc. buffered solutions. Ringer or Ringer-Lactate solution is particularly preferred as a liquid basis.

The phrase “administered in combination” as used herein refers to a situation, where the polymeric carrier cargo complex is administered to a subject before, concomittantly or after the administration of the second nucleic acid molecule encoding a protein or peptide to the same subject. Preferably, the time interval between the administration of the polymeric carrier cargo complex and the at least one second nucleic acid molecule, preferably an RNA, encoding a protein or peptide is less than about 48 hours, more preferably less than about 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, 1 hour, most preferably less than about 30 minutes, 15 minutes or 5 minutes. In a particularly preferred embodiment, the phrase “administered in combination” refers to concomitant administration of the polymeric carrier cargo complex and the at least one second nucleic acid molecule, i.e. the simultaneous administration of both components or the administration of both components within a time frame that typically comprises less than 5 minutes. The phrase “administered in combination” does not only refer to a situation, where the pharmaceutical carrier cargo complex is in physical contact with the at least one second nucleic acid molecule or formulated together with said second nucleic acid molecule. The phrase “administered in combination” as used herein comprises also the separate administration of the polymeric carrier cargo complex and the second nucleic acid molecule (e.g. by two separate injections), as long as the time interval between the two administrations does not exceed the interval as defined above. Alternatively, the polymeric carrier cargo complex and the second nucleic acid molecule may be administered in combination by mixing the polymeric carrier cargo complex and the second nucleic acid molecule prior to administration and administering the mixture to a subject. When the polymeric carrier cargo complex is formulated together with the second nucleic acid molecule or when a composition as defined herein is used, the polymeric carrier cargo complex and the second nucleic acid molecule may further, independently from each other, administered in combination via any of the administration routes as described herein.

The polymeric carrier cargo complex comprises as a cargo at least one nucleic acid molecule. In the context of the present invention, such a nucleic acid molecule may be any suitable nucleic acid, selected e.g. from any (single-stranded or double-stranded) DNA, preferably, without being limited thereto, e.g. genomic DNA, single-stranded DNA molecules, double-stranded DNA molecules, coding DNA, DNA primers, DNA probes, immunostimulatory/immunostimulating DNA, a (short) DNA oligonucleotide ((short) oligodesoxyribonucleotides), viral DNA, or may be selected e.g. from any PNA (peptide nucleic acid) or may be selected e.g. from any (single-stranded or double-stranded) RNA, preferably, without being limited thereto, a (short) RNA oligonucleotide ((short) oligoribonucleotide), a coding RNA, a messenger RNA (mRNA), a viral RNA, replicons, an immunostimulatory/immunostimulating RNA, a small interfering RNA (siRNA), an antisense RNA, a micro RNA, a small nuclear RNA (snRNA), a small-hairpin (sh) RNA or riboswitches, ribozymes or aptamers; etc. The nucleic acid molecule of the polymeric carrier cargo complex may also be a ribosomal RNA (rRNA), a transfer RNA (tRNA), a messenger RNA (mRNA), or a viral RNA (vRNA). Preferably, the nucleic acid molecule of the polymeric carrier cargo complex is an RNA. More preferably, the nucleic acid molecule of the polymeric carrier cargo complex is a (linear) single-stranded RNA, even more preferably an mRNA or an immunostimulatory/immunostimulating RNA.

Furthermore, the nucleic acid of the polymeric carrier cargo complex may be a single- or a double-stranded nucleic acid molecule or a partially double-stranded or partially single stranded nucleic acid, which are at least partially self complementary (both of these partially double-stranded or partially single stranded nucleic acid molecules are typically formed by a longer and a shorter single-stranded nucleic acid molecule or by two single stranded nucleic acid molecules, which are about equal in length, wherein one single-stranded nucleic acid molecule is in part complementary to the other single-stranded nucleic acid molecule and both thus form a double-stranded nucleic acid molecule in this region, i.e. a partially double-stranded or partially single stranded nucleic acid molecule. Preferably, the nucleic acid molecule may be a single-stranded nucleic acid molecule. Furthermore, the nucleic acid molecule may be a circular or linear nucleic acid molecule, preferably a linear nucleic acid molecule.

According to one alternative, the nucleic acid molecule of the inventive polymeric carrier cargo complex may be a coding nucleic acid, e.g. a DNA or RNA. Moreover, the polymeric carrier cargo complex may be administered in combination with at least one second nucleic acid molecule, which encodes a protein or a peptide.

According to one embodiment, the at least one first nucleic acid molecule and the at least one second nucleic acid molecule are both coding nucleic acid molecules. Preferably, the at least one first and the at least one second nucleic acid molecule each encode a different peptide or protein. In one embodiment, the first nucleic acid molecule has a sequence, which is distinct from the sequence of the second nucleic acid molecule, which is administered in combination with the polymeric carrier cargo complex. Alternatively, the first nucleic acid molecule and the second nucleic acid molecule may comprise the same sequence or be identical.

In the case of the at least one first nucleic acid molecule and/or of the second nucleic acid molecule, such a coding DNA or RNA may be any DNA or RNA as defined herein. Preferably, such a coding DNA or RNA may be a single- or a double-stranded DNA or RNA, more preferably a single-stranded DNA or RNA, and/or a circular or linear DNA or RNA, more preferably a linear DNA or RNA. Furthermore such a coding DNA or RNA may be a genomic DNA, a viral RNA or DNA, a replicon, a plasmid DNA or an mRNA. Even more preferably, the coding DNA or RNA may be a (linear) single-stranded DNA or RNA. Most preferably, the nucleic acid molecule according to the present invention may be a linear single-stranded messenger RNA (mRNA). Such an mRNA may occur as a mono-, di-, or even multicistronic RNA, i.e. an RNA which carries the coding sequences of one, two or more proteins or peptides. Such coding sequences in di-, or even multicistronic mRNA may be separated by at least one IRES sequence, e.g. as defined herein.

In a preferred embodiment, the at least one second nucleic acid molecule encodes a therapeutically active protein or an antigen as defined herein, preferably as disclosed in the context of “coding RNA”. In a particularly preferred embodiment, the at least one second nucleic acid molecule, which is administered in combination with the polymeric carrier cargo complex, encodes a peptide or a protein, which is capable of eliciting an immune response, preferably an adaptive immune response, after administration, especially intratumoral administration, to a host. Alternatively, the at least one second nucleic acid molecule encodes at least one therapeutically active peptide or protein, preferably selected from the group consisting of cytokines, chemokines, suicide gene products, immunogenic proteins or peptides, apoptosis inducers, angiogenesis inhibitors, heat shock proteins, tumor antigens, β-catenin inhibitors, activators of the STING pathway, checkpoint modulators, innate immune activators, antibodies, dominant negative receptors and decoy receptors, inhibitors of myeloid derived suppressor cells (MDSCs), IDO pathway inhibitors, and proteins or peptides that bind inhibitors of apoptosis.

In a particular embodiment, the first nucleic acid molecule of the herein defined polymeric carrier cargo complex and/or the second nucleic acid molecule administered in combination with the polymeric carrier cargo complex may contain backbone modifications, sugar modifications or base modifications. A backbone modification in connection with the present invention is a modification in which phosphates of the backbone of the nucleotides contained in the nucleic acid molecule of the inventive polymeric carrier cargo complex are chemically modified. A sugar modification in connection with the present invention is a chemical modification of the sugar of the nucleotides of the first nucleic acid molecule of the inventive polymeric carrier cargo complex and/or of the second nucleic acid molecule administered in combination with the polymeric carrier cargo complex. Furthermore, a base modification in connection with the present invention is a chemical modification of the base moiety of the nucleotides of the nucleic acid molecule of the inventive polymeric carrier cargo complex and/or of the second nucleic acid molecule administered in combination with the polymeric carrier cargo complex. Such modifications are disclosed above in the context of “RNA modifications”.

According to a further embodiment, the first nucleic acid molecule of the herein defined polymeric carrier cargo complex and/or the second nucleic acid molecule administered in combination with the polymeric carrier cargo complex can contain a lipid modification. Such a lipid-modified nucleic acid typically comprises a nucleic acid as defined herein. Such a lipid-modified first nucleic acid molecule of the polymeric carrier cargo complex or a lipid-modified second nucleic acid molecule administered in combination with the polymeric carrier cargo complex typically further comprises at least one linker covalently linked with that nucleic acid molecule, and at least one lipid covalently linked with the respective linker. Alternatively, the lipid-modified nucleic acid molecule comprises at least one nucleic acid molecule as defined herein and at least one (bifunctional) lipid covalently linked (without a linker) with that nucleic acid molecule. According to a third alternative, the lipid-modified nucleic acid molecule comprises a nucleic acid molecule as defined herein, at least one linker covalently linked with that nucleic acid molecule, and at least one lipid covalently linked with the respective linker, and also at least one (bifunctional) lipid covalently linked (without a linker) with that nucleic acid molecule.

According to a further preferred embodiment, the at least one RNA of the inventive composition is complexed with lipids to form one or more liposomes, lipoplexes, or lipid nanoparticles. Therefore, in one embodiment, the inventive composition comprises liposomes, lipoplexes, and/or lipid nanoparticles comprising the at least one RNA.

Lipid-based formulations have been increasingly recognized as one of the most promising delivery systems for RNA due to their biocompatibility and their ease of large-scale production. Cationic lipids have been widely studied as synthetic materials for delivery of RNA. After mixing together, nucleic acids are condensed by cationic lipids to form lipid/nucleic acid complexes known as lipoplexes. These lipid complexes are able to protect genetic material from the action of nucleases and deliver it into cells by interacting with the negatively charged cell membrane. Lipoplexes can be prepared by directly mixing positively charged lipids at physiological pH with negatively charged nucleic acids.

Conventional liposomes consist of a lipid bilayer that can be composed of cationic, anionic, or neutral (phospho)lipids and cholesterol, which encloses an aqueous core. Both the lipid bilayer and the aqueous space can incorporate hydrophobic or hydrophilic compounds, respectively. Liposome characteristics and behaviour in vivo can be modified by addition of a hydrophilic polymer coating, e.g. polyethylene glycol (PEG), to the liposome surface to confer steric stabilization. Furthermore, liposomes can be used for specific targeting by attaching ligands (e.g., antibodies, peptides, and carbohydrates) to its surface or to the terminal end of the attached PEG chains (Front Pharmacol. 2015 Dec. 1; 6:286).

Liposomes are colloidal lipid-based and surfactant-based delivery systems composed of a phospholipid bilayer surrounding an aqueous compartment. They may present as spherical vesicles and can range in size from 20 nm to a few microns. Cationic lipid-based liposomes are able to complex with negatively charged nucleic acids via electrostatic interactions, resulting in complexes that offer biocompatibility, low toxicity, and the possibility of the large-scale production required for in vivo clinical applications. Liposomes can fuse with the plasma membrane for uptake; once inside the cell, the liposomes are processed via the endocytic pathway and the genetic material is then released from the endosome/carrier into the cytoplasm. Liposomes have long been perceived as drug delivery vehicles because of their superior biocompatibility, given that liposomes are basically analogs of biological membranes, and can be prepared from both natural and synthetic phospholipids (Int J Nanomedicine. 2014; 9: 1833-1843).

Cationic liposomes have been traditionally the most commonly used non-viral delivery systems for oligonucleotides, including plasmid DNA, antisense oligos, and siRNA/small hairpin RNA-shRNA). Cationic lipids, such as DOTAP, (1,2-dioleoyl-3-trimethylammonium-propane) and DOTMA (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methyl sulfate) can form complexes or lipoplexes with negatively charged nucleic acids to form nanoparticles by electrostatic interaction, providing high in vitro transfection efficiency. Furthermore, neutral lipid-based nanoliposomes for RNA delivery as e.g. neutral 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC)-based nanoliposomes were developed. (Adv Drug Deliv Rev. 2014 February; 66: 110-116.).

Therefore, in one embodiment the at least one RNA of the inventive composition is complexed with cationic lipids and/or neutral lipids and thereby forms liposomes, lipid nanoparticles, lipoplexes or neutral lipid-based nanoliposomes.

Preferred cationic or polycationic compounds, which can be used as transfection or complexation agent may include cationic polysaccharides, for example chitosan, polybrene, cationic polymers, e.g. polyethyleneimine (PEI), cationic lipids, e.g. DOTMA: [1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE: Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS: Dioctadecylamidoglicylspermin, DIMRI: Dimyristo-oxypropyl dimethyl hydroxyethyl ammonium bromide, DOTAP: dioleoyloxy-3-(trimethylammonio)propane, DC-6-14: O,O-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride, CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium chloride, CLIP6: rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]trimethylammonium, CLIP9: rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium, oligofectamine, or cationic or polycationic polymers, e.g. modified polyaminoacids, such as β-aminoacid-polymers or reversed polyamides, etc., modified polyethylenes, such as PVP (poly(N-ethyl-4-vinylpyridinium bromide)), etc., modified acrylates, such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc., modified amidoamines such as pAMAM (poly(amidoamine)), etc., modified polybetaaminoester (PBAE), such as diamine end modified 1,4 butanediol diacrylate-co-5-amino-1-pentanol polymers, etc., dendrimers, such as polypropylamine dendrimers or pAMAM based dendrimers, etc., polyimine(s), such as PEI: poly(ethyleneimine), poly(propyleneimine), etc., polyallylamine, sugar backbone based polymers, such as cyclodextrin based polymers, dextran based polymers, chitosan, etc., silan backbone based polymers, such as PMOXA-PDMS copolymers, etc., blockpolymers consisting of a combination of one or more cationic blocks (e.g. selected from a cationic polymer as mentioned above) and of one or more hydrophilic or hydrophobic blocks (e.g. polyethyleneglycole); etc.

Additional Pharmaceutically Active Compounds:

Furthermore the inventive composition may comprise at least one additional pharmaceutically active component/compound. Alternatively or in addition to that, the at least one additional pharmaceutically active component/compound may be co-administered concomitant to the composition according to the invention. Therefore, the at least one additional pharmaceutically active component/compound may be administered in combination with the at least one RNA of the inventive composition or with the RNA containing composition according to the invention.

The phrases “administered in combination”, co-administration or “concomitant administration” as used herein refers to a situation, where the inventive composition or an ingredient thereof is administered to a subject before, concomittantly or after the administration of a further pharmaceutically active component to the same subject. The time interval between the administration of the inventive composition or an ingredient thereof and the at least one second pharmaceutically active component depends on the nature and biological effect of the particular pharmaceutically active component and can be determined by a physician. Preferably the time interval is less than about 48 hours, more preferably less than about 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, 1 hour, most preferably less than about 30 minutes, 15 minutes or 5 minutes. In a particularly preferred embodiment, the phrase “administered in combination” refers to concomitant administration of the inventive composition or an ingredient thereof and the at least one second pharmaceutically active component, i.e. the simultaneous administration of both compounds or the administration of both compounds within a time frame that typically comprises less than 5 minutes. The phrase “administered in combination” does not only refer to a situation, where the inventive composition or an ingredient thereof is in physical contact with the at least one second pharmaceutically active component or formulated together with said second pharmaceutically active component. The phrase “administered in combination” as used herein comprises also the separate administration of the inventive composition or an ingredient thereof and the second pharmaceutically active component (e.g. by two separate injections). Alternatively, the inventive composition or an ingredient thereof and the second pharmaceutically active component may be administered in combination by mixing the inventive composition or an ingredient thereof and the second pharmaceutically active component prior to administration and administering the mixture to a subject. When the inventive composition or an ingredient thereof is formulated together with the second pharmaceutically active component or when a composition as defined herein is used, the inventive composition or an ingredient thereof and the second pharmaceutically active component may further, independently from each other, administered in combination via any of the administration routes as described herein.

A pharmaceutically active component/compound in this connection is a compound that has a therapeutic effect to heal, ameliorate or prevent a particular indication or disease, namely a tumor or cancer disease. Such compounds include, without implying any limitation, peptides or proteins, preferably as defined herein, nucleic acids, preferably as defined herein, (therapeutically active) low molecular weight organic or inorganic compounds (molecular weight less than 5000, preferably less than 1000), sugars, antigens or antibodies, preferably as defined herein, therapeutic agents already known in the prior art, antigenic cells, antigenic cellular fragments, cellular fractions, cell wall components (e.g. polysaccharides), modified, attenuated or de-activated (e.g. chemically or by irradiation) pathogens (virus, bacteria etc.), adjuvants, etc.

In a preferred embodiment, the inventive composition additionally comprises at least one further pharmaceutically active component/compound, wherein the at least one additional pharmaceutically active component is selected from cytokines, chemokines, suicide gene products, immunogenic proteins or peptides, apoptosis inducers, angiogenesis inhibitors, heat shock proteins, tumor antigens, β-catenin inhibitors, activators of the STING pathway, checkpoint modulators, innate immune activators, antibodies, dominant negative receptors and decoy receptors, inhibitors of myeloid derived suppressor cells (MDSCs), IDO pathway inhibitors, proteins or peptides that bind inhibitors of apoptosis, anti-bacterial agents, anti-viral agents, adjuvants, chemotherapeutic agents and kinase inhibitors.

Alternatively, or in addition to that, the at least one additional pharmaceutically active component may be co-administered concomitant to the at least one RNA of the RNA containing composition or the inventive composition or may be used in combination with the at least one RNA of the RNA containing composition or the inventive composition.

In this context protein-based cytokines, chemokines, suicide gene products, immunogenic proteins or peptides, apoptosis inducers, angiogenesis inhibitors, heat shock proteins, tumor antigens, β-catenin inhibitors, activators of the STING pathway, checkpoint modulators, innate immune activators, antibodies, dominant negative receptors and decoy receptors, inhibitors of myeloid derived suppressor cells (MDSCs), IDO pathway inhibitors, and proteins or peptides that bind inhibitors of apoptosis or fragments and variants thereof as disclosed above in the context of “coding RNA” may be used as additional pharmaceutically active component. Alternatively, nucleic acids encoding these proteins or fragments or variants thereof may be used as additional pharmaceutically active component.

1. Cytokines:

In this context protein-based cytokines, or fragments and variants thereof as disclosed above in the context of “coding RNA” may be used as additional pharmaceutically active component. Alternatively, nucleic acids encoding these proteins or fragments or variants thereof may be used as additional pharmaceutically active component.

Preferably the cytokine is an interleukin (IL). One or more interleukins may be chosen e.g. from the following list: IL-1α, IL-1β, IL-1ra (antagonist), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10; IL-11, IL-12, IL-13, IL14, IL-15, IL-16, IL-17A, IL-17B, EL-17C, IL-17D, IL-17E, IL-17F, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28A/B, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35. Moreover the cytokine may be one or more cytokines chosen from the TNF family, e.g. chosen from the following list: TNF, especially TNFα, LTα, LTβ, LIGHT, TWEAK, APRIL, BAFF, TL1A, GITRL, OX40L, CD40L (CD154), FASL, CD27L, CD30L, 4-1BBL, TRAIL, RANK ligand. Further examples of preferred cytokines may be chosen from the following list: FLT3 ligand, G-CSF, GM-CSF, IFNα/β/ω, IFNγ, LIF, M-CSF, MIF, OSM, Stem Cell Factor, TGFβ1, TGFβ2, TGFβ3, TSLP ligand.

Particularly preferred are cytokines chosen from the following list: IL-12, IL-15, IL-2, IFNγ, TNFα, IL-18, IFNα, IL-1β, IL-32, IL-7, IL-21, IL-8, GM-CSF.

In this context particularly preferred are cytokines as disclosed in Table 1 above.

In a particularly preferred embodiment, the at least one RNA of the RNA containing composition is an immunostimulating RNA, preferably according to SEQ ID NOs. 5, 394, or 10072, and is combined with at least one cytokine as defined above, preferably IL-2, IL-12, CD40L or IL-15 or a fragment or variant thereof.

1. Chemokines

In this context protein-based chemokines, or fragments and variants thereof as disclosed above in the context of “coding RNA” may be used as additional pharmaceutically active component. Alternatively, nucleic acids encoding these proteins or fragments or variants thereof may be used as additional pharmaceutically active component.

Preferred chemokines may be chosen from the following list: CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, XCL1, XCL2, CX3CL1.

In this context particularly preferred are chemokines as disclosed in Table 2 above.

In a particularly preferred embodiment, the at least one RNA of the RNA containing composition is an immunostimulating RNA, preferably according to SEQ ID NOs. 5, 394, or 10072 and is combined with at least one chemokine as defined above or a fragment or variant thereof.

2. Suicide Enzymes

In this context protein-based suicide enzymes, or fragments and variants thereof as disclosed above in the context of “coding RNA” may be used as additional pharmaceutically active component. Alternatively, nucleic acids encoding these proteins or fragments or variants thereof may be used as additional pharmaceutically active component.

The suicide enzyme is preferably a nucleotide metabolizing enzyme. Preferably the suicide enzyme is used in combination with a prodrug which is a substrate of the suicide enzyme, and which is converted to a cytotoxic compound by the suicide enzyme. One or more preferred suicide enzymes may be chosen from the following list: thymidine kinase, preferably a viral thymidine kinase, more preferrably Herpes simplex virus thymidine kinase, Varicella zoster thymidine kinase; a plant thymidine kinase, preferably a tomato thymidine kinase; cytosine deaminase, preferably bacterial cytosine deaminase or Yeast cytosine deaminase; deoxynucleoside kinase, preferably Drosophila melanogaster deoxynucleoside kinase; deoxycytidine kinase, preferably a mammalian deoxycytidine kinase, purine nucleoside phosphorylase, preferably a bacterial purine nucleoside phosphorylase.

In this context particularly preferred are suicide enzymes (suicide gene products) as disclosed in Table 3 and 4 above.

In a particularly preferred embodiment, the at least one RNA of the RNA containing composition is an immunostimulating RNA, preferably according to SEQ ID NOs. 5, 394, or 10072 and is combined with at least one suicide enzyme as defined above or a fragment or variant thereof.

3. Immunogenic Proteins or Peptides

In this context protein-based immunogenic proteins or peptides, or fragments and variants thereof as disclosed above in the context of “coding RNA” may be used as additional pharmaceutically active component. Alternatively, nucleic acids encoding these proteins or peptides or fragments or variants thereof may be used as additional pharmaceutically active component.

The immunogenic protein or peptide is preferably a pathogenic antigen to utilize preexisting immunity against such antigens for treatment of tumor and/or cancer diseases. The memory immune response is triggered and the immune system is strengthened for attacking tumor cells.

Preferred examples of immunogenic proteins or peptides for this embodiment of the invention are proteins or peptides of widespread pathogens, i.e. pathogens with which every organism, in particular mammals, preferably humans, has a high probability of being infected at least once in his/her lifetime. These include, for example, any structural or non-structural protein or peptide of:

-   -   influenza virus type A or B or any other orthomyxovirus         (influenza type C),     -   picornaviruses, such as rhinovirus or hepatitis A virus,     -   togaviruses, such as alphavirus or rubivirus, e.g. Sindbis,         Semliki-Forest or rubeolavirus (measles virus),     -   rubella virus (German measles virus),     -   coronaviruses, in particular subtypes HCV-229E or HCV-0C43,     -   rhabdoviruses, such as rabies virus,     -   paramyxoviruses, such as mumps virus,     -   reoviruses, such as group A, B or C rotavirus,     -   hepadnaviruses, such as hepatitis B virus,     -   papoviruses, such as human papillomaviruses (HPV) of any         serotype, especially from 1 to 75,     -   adenoviruses, in particular type 1 to 47,     -   herpesviruses, such as Herpes simplex virus 1, 2 or 3,     -   cytomegalovirus (CMV), preferably CMVpp65,     -   Epstein Barr virus (EBV),     -   vaccinia viruses and     -   the bacterium Chiamydophila pneumoniae (Chlamydia pneumoniae).

Further examples of preferred immunogenic proteins or peptides are proteins or peptides of pathogens which only seldom infect an organism. These proteins or peptide include, for example, any structural or non-structural protein or peptide of:

-   -   Flaviviruses, such as dengue virus type 1 to 4, yellow fever         virus, West Nile virus, Japanese encephalitis virus     -   hepatitis C virus,     -   caliciviruses,     -   filoviruses, such as Ebola virus,     -   bornaviruses,     -   bunyaviruses, such as Rift Valley fever virus,     -   arenaviruses, such as LCMV (lymphocytic choriomeningitis virus)         or hemorrhagic fever viruses,     -   retroviruses, such as HIV and     -   parvoviruses.

In a particularly preferred embodiment, the at least one RNA of the RNA containing composition is an immunostimulating RNA, preferably according to SEQ ID NOs. 5, 394, or 10072 and is combined with at least one immunogenic protein or peptide as defined above, preferably influenza nucleoprotein (NP) or a fragment or variant thereof.

4. Apoptosis Inducers:

In this context protein-based apoptosis inducers, or fragments and variants thereof as disclosed above in the context of “coding RNA” may be used as additional pharmaceutically active component. Alternatively, nucleic acids encoding these proteins or peptides or fragments or variants thereof may be used as additional pharmaceutically active component.

Preferably, an apoptosis inducer is chosen from the group consisting of the Bcl-2 family and tumor suppressor protein p53 and ligands of transmembrane death receptors, especially the TNF (tumor necrosis factor) receptor gene superfamily, pro-apoptic receptor agonists and Beclin-1.

A particularly preferred apoptosis inducer in the context of the present invention is Beclin-1 (derived from the BECN1 gene).

Further preferred examples of apoptosis inducers may be chosen from the following list: Bcl-10, Bax, Bak, Bid, Bad, Bim, Bik, Blk, Cytochrome c, Caspases, especially Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Death domain, especially of Fas, preferably FasL, TNFα, Apo2L/TRAIL, agonist of DR4 and/or DR5, Apo3L, DR4 agonistic antibody, DR5 agonistic antibody, protein kinase R (PKR) (preferably constitutive active PKR), Granzyme B.

In this context particularly preferred are apoptosis inducers as disclosed in Table 5 and 6 above.

In a particularly preferred embodiment, the at least one RNA of the RNA containing composition is an immunostimulating RNA, preferably according to SEQ ID NOs. 5, 394, or 10072 and is combined with at least one apoptosis inducer as defined above, or a fragment or variant thereof.

5. Angiogenesis Inhibitors

In this context protein-based angiogenesis inducers, or fragments and variants thereof as disclosed above in the context of “coding RNA” may be used as additional pharmaceutically active component. Alternatively, nucleic acids encoding these proteins or peptides or fragments or variants thereof may be used as additional pharmaceutically active component.

Preferred examples of angiogenesis inhibitors according to the invention may be chosen from the following list: interferon alpha (IFN-α), (interferon beta) IFN-β, interferon gamma (IFN-γ), CXCL9, CXCL10, interleukin 12 (IL-12), platelet factor 4 (PF-4), tumor necrosis factor alpha (TNF-α), soluble fms-like tyrosine kinase 1 (sFLT-1), Fetal Liver Kinase 1 (FLK-1), Angiostatin, Endostatin, Vasostatin, Canstatin, Tumstatin, 16 kD prolacin fragment, tissue inhibitor of metalloproteinases 1 (TIMP-1), tissue inhibitor of metalloproteinases 2 (TIMP-2), tissue inhibitor of metalloproteinases 3 (TIMP-3), thrombospondin 1 (TSP-1), thrombospondin 2 (TSP-2), Maspin, PEX, soluble Tyrosine-protein kinase receptor 1 (sTie1), soluble Angiopoietin-1 receptor 2 (sTie2), Angiopoietin-1, Angiopoietin-2, Antivascular endothelial growth factor receptor 2 (VEGFR2) antibody (e.g. Alacizumab, Ramucirumab), Anti-vascular endothelial growth factor (VEGF) antibody (e.g. Brolucizumab, Ranibizumab, Bevacizumab), and Anti-vascular endothelial growth factor receptor 1 (VEGFR1) antibody (e.g. Icrucumab).

In this context particularly preferred are angiogenesis inhibitors as disclosed in Table 7 above.

In a particularly preferred embodiment, the at least one RNA of the RNA containing composition is an immunostimulating RNA, preferably according to SEQ ID NOs. 5, 394, or 10072 and is combined with at least one angiogenesis inhibitor as defined above, or a fragment or variant thereof.

6. Heat Shock Proteins:

In this context protein-based heat-shock proteins, or fragments and variants thereof as disclosed above in the context of “coding RNA” may be used as additional pharmaceutically active component. Alternatively, nucleic acids encoding these proteins or peptides or fragments or variants thereof may be used as additional pharmaceutically active component.

Preferably, the heat shock protein may be chosen from the following list: HSP27, HSP47 (serpin H1), HSP60, HSP70, HSC70, GRP78 (BiP), HSP90, HSP110, GRP94 (gp96), GRP170 (ORP150), PDI/PDIA, CRT/CALR.

In this context particularly preferred are heat shock proteins as disclosed in Table 8 above.

In a particularly preferred embodiment, the at least one RNA of the RNA containing composition is an immunostimulating RNA, preferably according to SEQ ID NOs. 5, 394, or 10072 and is combined with at least one heat shock protein as defined above, or a fragment or variant thereof.

7. Tumour Antigens:

In this context protein-based tumor antigens, or fragments and variants thereof as disclosed above in the context of “coding RNA” may be used as additional pharmaceutically active component. Alternatively, nucleic acids encoding these proteins or peptides or fragments or variants thereof may be used as additional pharmaceutically active component.

In this context particularly preferred are tumor antigens as disclosed in Table 9 above.

In a particularly preferred embodiment, the at least one RNA of the RNA containing composition is an immunostimulating RNA, preferably according to SEQ ID NOs. 5, 394, or 10072 and is combined with at least one tumor antigen as defined above, or a fragment or variant thereof.

8. β-Catenin Inhibitors:

In this context protein-based β-catenin inhibitors, or fragments and variants thereof as disclosed above in the context of “coding RNA” may be used as additional pharmaceutically active component. Alternatively, nucleic acids encoding these proteins or peptides or fragments or variants thereof may be used as additional pharmaceutically active component.

Particular preferred β-catenin inhibitors according to the present invention comprise TAT-NLS-BLBD-6, axin-1, TCF-4, GSK-3b, DKK-1, Dvl-1 derivatives or fragments thereof.

Chemical β-Catenin Inhibitors:

According to the present invention, the at least one additional active pharmaceutical ingredient which may be contained in the inventive composition, and/or which may be co-administered, or which may be combined with the inventive composition may be a chemical β-catenin inhibitors. Chemical β-catenin inhibitors are known in the art that may be administered according to the present invention. Preferably the chemical β-catenin inhibitor is chosen from the following list: PKF118-310, CGP049090, PKF115-584, PKF222-815, PKF118-744, ICG001, CCT036477, XAV939, acyl hydrazones (HQBA), molecules with 2,3,6-trisubstituted pyrido[2,3,-b]pyrazine core skeletons, carnosic acid, CCT031374, iCRT-3,5,14, NC043, Ibuprofin, aspirin.

The following table 13 summarizes examples of small molecular inhibitors of β-catenin signaling which are particularly preferred in this context.

TABLE 13 β-catenin inhibitors Inhibitor Target Reference PKF118-310, CGP049090, PKF115- beta-catenin—TCF Lepourcelet et al., 2004. 584, PKF222-815 and PKF118-744 interaction Cancer Cell 5:91-102 ICG001 beta-catenin—CBP Emami et al., 2004. Proc Natl interaction Acad Sci USA 101:12682-7 CCT036477 beta-catenin—TCF Ewan et al., 2010. Cancer interaction Res. 70:5963-73 XAV939 Tankyrase Huang et al., 2009. Nature 461:614-20 acyl hydrazones (HQBA) Iron chelators Song et al., 2011. Cancer Res. 71:7628-39; Coombs et al., 2012. Oncogene 31:213-25 molecules with 2,3,6-trisubstituted beta-catenin Gong et al., 2011. Bioorg pyrido[2,3,-b] pyrazine core Med Chem. 19:5639-47 skeletons carnosic acid beta-catenin/BCL9 de la Roche et al., Nat Commun. 3:680 CCT031374 beta-catenin Thorne et al., 2010. Nat Chem Biol. 6:829-36 iCRT-3,5,14, NC043 beta-catenin—TCF Wang et al., 2011. Cell Res. interaction 21:730-40; Gonsalves et al., 2011. Proc Natl Acad Sci USA 108:5954-63 Ibuprofin, aspirin Cox2 Inhibitors Greenspan et al., 2011. Cancer Prev Res. 4:161-71

In a particularly preferred embodiment, the at least one RNA of the RNA containing composition is an immunostimulating RNA, preferably according to SEQ ID NOs. 5, 394, or 10072 and is combined with at least one β-catenin inhibitor as defined above, or a fragment or variant thereof.

9. Activators of the STING Pathway

In this context protein-based activators of the STING pathway, or fragments and variants thereof as disclosed above in the context of “coding RNA” may be used as additional pharmaceutically active component. Alternatively, nucleic acids encoding these proteins or peptides or fragments or variants thereof may be used as additional pharmaceutically active component. Preferably, the at least one activator (stimulator) of the STING pathway is chosen from an activating protein or a constitutively active protein of the STING pathway, preferably DDX41, STING, cGAS, IRF3, TBK1 or STAT6 or a fragment or variant thereof.

Chemical STING-Pathway Activators:

In a further preferred embodiment the optional additional pharmaceutically active component may be selected from chemical activators of the STING pathway which are preferably selected from cyclic dinucleotides and xanthenone analogs.

Table 14 shows examples of chemical STING agonists. Further examples of STING agonists are disclosed in WO2014189805.

TABLE 14 Activators of STING pathway Class of STING activator examples cyclic dinucleotides 3′3′-cGAMP, 2′3′-cGAMP, 2′2′-cGAMP, c-di-APM, c-di-GMP, c-di-IMP, c-di-UMP xanthenone analogs DMXAA, 5,6-dimethylxanthenone-4-acetic acid

In a particularly preferred embodiment, the at least one RNA of the RNA containing composition is an immunostimulating RNA, preferably according to SEQ ID NOs. 5, 394, or 10072 and is combined with at least one STING pathway activator as defined above, or a fragment or variant thereof.

10. Checkpoint Modulators

In this context protein-based checkpoint modulators, or fragments and variants thereof as disclosed above in the context of “coding RNA” may be used as additional pharmaceutically active component. Alternatively, nucleic acids encoding these proteins or peptides or fragments or variants thereof may be used as additional pharmaceutically active component.

In preferred embodiments of the present invention the checkpoint modulator is a modulator of B7-1/CD80, B7-2/CD86, B7-H1/PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7/HHLA2, BTLA, CD28, CD28H/IGPR-1, CTLA-4, ICOS, PD-1, PD-L2/B7-DC, PDCD6, VISTA/B7-H5/PD-1H, BTN1A1/Butyrophilin, BTN2A1, BTN2A2/Butyrophilin 2A2, BTN3A1/2, BTN3A2, BTN3A3, BTNL2/Butyrophilin-like 2, BTNL3, BTNL4, BTNL6, BTNL8, BTNL9, BTNL10, CD277/BTN3A1, LAIR1, LAIR2, CD96, CD155/PVR, CRTAM, DNAM-1/CD226, Nectin-2/CD112, Nectin-3, TIGIT, LILRA3/CD85e, LILRA4/CD85g/ILT7, LILRB1/CD85j/ILT2, LILRB2/CD85d/ILT4, LILRB3/CD85a/ILT5, LILRB4/CD85k/ILT3, 4-1BB/TNFRSF9/CD137, 4-1BB Ligand/TNFSF9, BAFF/BLyS/TNFSF13B, BAFF R/TNFRSF13C, CD27/TNFRSF7, CD27 Ligand/TNFSF7, CD30/TNFRSF8, CD30 Ligand/TNFSF8, CD40/TNFRSF5, CD40 Ligand/TNFSF5, DR3/TNFRSF25, GITR/TNFRSF18, GITR Ligand/TNFSF18, HVEM/TNFRSF14, LIGHT/TNFSF14, Lymphotoxin-alpha/TNF-beta, OX40/TNFRSF4, OX40 Ligand/TNFSF4, RELT/TNFRSF19L, TACI/TNFRSF13B, TL1A/TNFSF15, TNF-alpha, TNF RII/TNFRSF1B, 2B4/CD244/SLAMF4, BLAME/SLAMF8, CD2, CD2F-10/SLAMF9, CD48/SLAMF2, CD58/LFA-3, CD84/SLAMF5, CD229/SLAMF3, CRACC/SLAMF7, NTB-A/SLAMF6, SLAM/CD150, TIM-1/KIM-1/HAVCR, TIM-3, TIM-4, CD7, CD96, CD160, CD200, CD300a/LMIR1, CRTAM, DAP12, Dectin-1/CLEC7A, DPPIV/CD26, EphB6, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM-1, LAG-3, TIM-1/KIM-1/HAVCR, TIM-4, TSLP R, or any combinations thereof.

Preferably, the checkpoint modulator is selected from agonistic antibodies, antagonistic antibodies, ligands, dominant negative receptors and decoy receptors or combinations thereof.

Preferably, the agonistic antibody is chosen from the following list: anti-4-1BB, anti-OX40, anti-GITR, anti-CD28, anti-CD27, anti-CD-40anti-ICOS, anti-TNFRSF25, and anti-LIGHT.

Preferably, the antagonistic antibody is chosen from the list of anti-CTLA4, anti-PD1, anti-PD-L1, anti-Vista, anti-Tim-3, anti-LAG-3, and anti-BTLA.

Particularly preferred are the anti-CTLA-4 antibodies ipilimumab (Yervoy®), tremelimumab, and AGEN-1884.

Particularly preferred are the anti-PD1 antibodies Nivolumab (MDX-1106/BMS-936558/ONO-4538), (Brahmer et al., 2010. J Clin Oncol. 28(19):3167-75; PMID: 20516446); Pidilizumab (CT-011), (Berger et al., 2008. Clin Cancer Res. 14(10):3044-51; PMID: 18483370); Pembrolizumab (MK-3475, SCH 900475); AMP-224, and MED10680 (AMP-514).

Particularly preferred are the anti-PD-L1 antibodies MDX-1105/BMS-936559 (Brahmer et al. 2012. N Engl J Med. 366(26):2455-65; PMID: 22658128); atezolizumab (MPDL3280A/RG7446); durvalumab (MED14736); and avelumab (MSB0010718).

According to the present invention checkpoint modulators according to Table 15 are particularly preferred:

TABLE 15 Antibodies directed against immune checkpoint proteins Name Target Urelumab 4-1BB/CD137 PF-05082566 4-1BB/CD137 8H9 B7-H3 Enoblituzumab B7-H3 Ipilimumab CD152/CTLA-4 Ticilimumab (=tremelimumab) CD152/CTLA-4 Tremelimumab CD152/CTLA-4 Varlilumab CD27 Teneliximab CD40 Vorsetuzumab mafodotin CD70 Lirilumab KIR2D GSK-3174998 OX40 MEDI-6469 OX40 MEDI-6383 OX40 MEDI-0562 OX40 PF-04518600 OX40 RG-7888 OX40 PF-06801591 PD-1 BGBA-317 PD-1 MEDI-0680 PD-1 MK-3475 PD-1 Nivolumab PD-1 PDR-001 PD-1 Pembrolizumab PD-1 Pidilizumab PD-1 REGN-2810 PD-1 SHR-1210 PD-1 TSR-042 PD-1 MDX-1106 PD-1 Merck 3745 PD-1 CT-011 PD-1 MEDI-0680 PD-1 PDR001 PD-1 REGN2810 PD-1 BGB-108 PD-1 BGB-A317 PD-1 AMP-224 PD-1 Atezolizumab PD-L1 (CD274) Avelumab PD-L1 (CD274) BMS-936559 PD-L1 (CD274) Durvalumab PD-L1 (CD274) MEDI-4736 PD-L1 (CD274) MPDL33280A PD-L1 (CD274) YW243.55.570 PD-L1 (CD274) MDX-1105 PD-L1 (CD274) MSB0010718C PD-L1 (CD274)

In a further preferred embodiment the checkpoint modulator is a decoy receptor (e.g. a soluble receptor). Preferably, the decoy receptor is a soluble PD1 receptor. In a particularly preferred embodiment the RNA sequence encoding a soluble PD1 receptor comprises an RNA sequence according to SEQ ID NO: 389

In a further preferred embodiment the checkpoint modulator is a ligand of an immune checkpoint protein. Preferably, the ligand is CD40 Ligand (CD40L).

In a particularly preferred embodiment, the at least one RNA of the RNA containing composition is an immunostimulating RNA, preferably according to SEQ ID NOs. 5, 394, or 10072 and is combined with at least one checkpoint modulator as defined above, preferably selected from an anti-CTLA4 antibody, an anti-PD1 antibody, an anti PD-L1 antibody, a CD40 ligand, or a soluble PD1 receptor, or a fragment or variant thereof.

11. Innate Immune Activators

In this context protein-based innate immune activators or fragments and variants thereof as disclosed above in the context of “coding RNA” may be used as additional pharmaceutically active component. Alternatively, nucleic acids encoding these proteins or peptides or fragments or variants thereof may be used as additional pharmaceutically active component.

In this context innate immune activators may be selected from mammalian, in particular human adjuvant proteins, which typically comprise any human protein or peptide, which is capable of eliciting an innate immune response (in a mammal), e.g. as a reaction of the binding of an exogenous TLR ligand to a TLR. More preferably, human adjuvant proteins are selected from the group consisting of proteins which are components and ligands of the signalling networks of the pattern recognition receptors including TLR, NLR and RLH, including TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11; NOD1, NOD2, NOD3, NOD4, NOD5, NALP1, NALP2, NALP3, NALP4, NALP5, NALP6, NALP6, NALP7, NALP7, NALP8, NALP9, NALP10, NALP11, NALP12, NALP13, NALP14, I IPAF, NAIP, CIITA, RIG-I, MDA5 and LGP2, the signal transducers of TLR signaling including adaptor proteins including e.g. Trif and Cardif; components of the Small-GTPases signalling (RhoA, Ras, Rac1, Cdc42, Rab etc.), components of the PIP signalling (PI3K, Src-Kinases, etc.), components of the MyD88-dependent signalling (MyD88, IRAK1, IRAK2, IRAK4, TIRAP, TRAF6 etc.), components of the MyD88-independent signalling (TICAM1, TICAM2, TRAF6, TBK1, IRF3, TAK1, IRAK1 etc.); the activated kinases including e.g. Akt, MEKK1, MKK1, MKK3, MKK4, MKK6, MKK7, ERK1, ERK2, GSK3, PKC kinases, PKD kinases, GSK3 kinases, JNK, p38MAPK, TAK1, IKK, and TAK1; the activated transcription factors including e.g. NF-κB, c-Fos, c-Jun, c-Myc, CREB, AP-1, Elk-1, ATF2, IRF-3, IRF-7.

Mammalian, in particular human adjuvant proteins may furthermore be selected from the group consisting of heat shock proteins, such as HSP10, HSP60, HSP65, HSP70, HSP75 and HSP90, gp96, Fibrinogen, TypIII repeat extra domain A of fibronectin; or components of the complement system including C1q, MBL, C1r, C1s, C2b, Bb, D, MASP-1, MASP-2, C4b, C3b, C5a, C3a, C4a, C5b, C6, C7, C8, C9, CR1, CR2, CR3, CR4, C1qR, C1INH, C4bp, MCP, DAF, H, I, P and CD59, or induced target genes including e.g. Beta-Defensin, cell surface proteins; or human adjuvant proteins including trif, flt-3 ligand, Gp96 or fibronectin, etc., or any species homolog of any of the above human adjuvant proteins. Furthermore HGMB1 may be used as adjuvant protein.

Mammalian, in particular human adjuvant proteins may furthermore comprise cytokines which induce or enhance an innate immune response, including IL-1 alpha, IL1 beta, IL-2, IL-6, IL-7, IL-8, IL-9, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21, IL-23, TNFalpha, IFNalpha, IFNbeta, IFNgamma, GM-CSF, G-CSF, M-CSF; chemokines including IL-8, IP-10, MCP-1, MIP-1alpha, RANTES, Eotaxin, CCL21; cytokines which are released from macrophages, including IL-1, IL-6, IL-8, IL-12 and TNF-alpha; as well as IL-1R1 and IL-1 alpha.

Therefore in this context it particularly preferred that the at least innate immune activator, is preferably an adjuvant protein, more preferably a human adjuvant protein, or a fragment or variant thereof.

In this context it is particularly preferred that I constitutive active variant of an adjuvant protein is used as innate immune activator, preferably a constitutive active variant of RIG-1 (ΔRIGI).

In another preferred embodiment the at least one innate immune activator is HGMB1, or a fragment or variant thereof.

In this context particularly preferred are innate immune activators as disclosed in Table 11 above.

In a particularly preferred embodiment, the at least one RNA of the RNA containing composition is an immunostimulating RNA, preferably according to SEQ ID NOs. 5, 394, or 10072 and is combined with at least one innate immune activator as defined above, or a fragment or variant thereof.

12. Antibodies, Decoy Receptors and Dominant Negative Receptors

In this context protein-based antibodies, decoy receptors, or dominant negative receptors or fragments and variants thereof as disclosed above in the context of “coding RNA” may be used as additional pharmaceutically active component. Alternatively, nucleic acids encoding these proteins or peptides or fragments or variants thereof may be used as additional pharmaceutically active component.

According to the present invention antibodies according to Table 16 are particularly preferred:

TABLE 16 Antibodies directed against proteins accociated with tumor or cancer development Name Target 3F8 GD2 Abagovomab CA-125 imitation Abciximab Platelet glycoprotein GPIIb/IIIa Adecatumumab EpCAM (CD326) Afutuzumab CD20 Alacizumab pegol VEGFR2 Alemtuzumab CD52 Altumomab pentetate CEA Amatuximab mesothelin Anatumomab mafenatox 5T4 Anetumab ravtansine mesothelin Apolizumab HLA-DR beta apomab TRAIL-R2 (CD262) Arcitumomab CEA Ascrinvacumab ACVRL1 Bavituximab phosphatidylserine Bectumomab CD22 Belimumab BAFF Besilesomab CEA Bevacizumab VEGF-A Bivatuzumab mertansine CD44v6 Blinatumomab CD19 × CD3 Brentuximab vedotin CD30 (TNFRSF8) Brontictuzumab NOTCH1 canakinumab IL-1β Cantuzumab mertansine CanAg Cantuzumab ravtansine MUC1 (CD227) Capromab pendetide PSMA Carlumab MCP-1 Catumaxomab EpCAM × CD3 cBR-doxorubicin immunoconjugate CD174 (Lewis Y) Cetuximab EGFR (HER1/ERBB1) Citatuzumab bogatox EpCAM Cixutumumab IGF-1R Clivatuzumab tetraxetan MUC1 (CD227) Codrituzumab glypican 3 Coltuximab ravtansine CD19 Conatumumab TRAIL-R2 (CD262) Dacetuzumab CD40 Dalotuzumab IGF-1R Dalotuzumab insulin-like growth factor I receptor Daratumumab CD38 (cyclic ADP ribose hydrolase) Demcizumab DLL4 Denintuzumab mafodotin CD19 Denosumab RANKL Depatuxizumab EGFR (HER1/ERBB1) Derlotuximab histone complex Detumomab unknown (B-lymphoma cells) Dinutuximab B4GALNT1 Drozitumab TRAIL-R2 (CD262) Duligotumab HER3 (ERBB3) Duligotuzumab EGFR (HER1/ERBB1) Dusigitumab ILGF2 Ecromeximab GD3 ganglioside Edrecolomab EpCAM Elgemtumab ERBB3 Elotuzumab SLAMF7 (CD319) Elsilimomab IL-6 Emactuzumab CSF1R Emibetuzumab HGFR Emibetuzumab MET Enavatuzumab TNFRSF12A Enfortumab vedotin AGS-22M6 Enoticumab DLL4 Ensituximab MUC5AC Epitumomab cituxetan MUC1 (CD227) Epratuzumab CD22 Ertumaxomab HER2 (ERBB2/neu) × CD3 Etaracizumab integrin α5β3 Faralimomab IFNA1 Farletuzumab FOLR1 alpha FBTA CD20 × CD3 Ficlatuzumab HGFR Figitumumab IGF-1R Flanvotumab TYRP1(glycoprotein 75) Fresolimumab TGF-β Futuximab EGFR (HER1/ERBB1) Galiximab CD80 Gantiumab IGF-1R Gemtuzumab ozogamicin CD33 Girentuximab Carbonic anhydrase 9 (CA9/CAIX) Glembatumumab vedotin GPNMB glycooptimized trastuzumab-GEX HER2 (ERBB2/neu) Ibritumomab tiuxetan CD20 Icrucumab VEGFR-1 Igovomab MUC16 IMAB362 Claudin-18 (CLDN18.2) Imgatuzumab EGFR (HER1/ERBB1) Indatuximab ravtansine SDC1 Indusatumab vedotin GUCY2C inebilizumab CD19 Inotuzumab ozogamicin CD22 Intetumumab CD51 Iratumumab CD30 (TNFRSF8) Isatuximab CD38 Labetuzumab CEA Lenzilumab CSF2 Lexatumumab TRAIL-R2 (CD262) Lifastuzumab vedotin NaPi2B Lilotomab satetraxetan CD37 Lintuzumab CD33 Lorvotuzumab mertansine CD56 Lucatumumab CD40 Lumiliximab CD23 (IgE receptor) Lumretuzumab ERBB3 Mapatumumab TRAIL-R1 (CD261) Margetuximab HER2 (ERBB2/neu) Matuzumab EGFR (HER1/ERBB1) Mepolizumab IL-5 Milatuzumab CD74 Minretumomab TAG-72 Mirvetuximab soravtansine FOLR1 alpha Mitumomab GD3 (ganglioside) Mogamulizumab CCR4 Moxetumomab pasudotox CD22 Nacolomab tafenatox C242 antigen Naptumomab estafenatox 5T4 Narnatumab RON Necitumumab EGFR (HER1/ERBB1) Nesvacumab ANGPT2 (angiopoietin 2) Nimotuzumab EGFR (HER1/ERBB1) Nofetumomab merpentan EpCAM binutuzumab CD20 Ocaratuzumab CD20 Ofatumumab CD20 Olaratumab PDGFRα Onartuzumab MET Ontuxizumab CD248 (TEM1) Oportuzumab monatox EpCAM Oregovomab CA-125 Otlertuzumab CD37 Panitumumab EGFR (HER1/ERBB1) Pankomab MUC1 (tumor specific glycosylation) Parsatuzumab EGFL7 Pasotuxizumab FOLH1 Patritumab HER3 (ERBB3) Pemtumomab MUC1 (CD227) Pertuzumab HER2 (ERBB2/neu) Pinatuzumab vedotin CD22 Pintumomab adenocarcinoma antigen Polatuzumab vedotin CD79B Racotumomab NGcGM3 Radretumab EDB (fibronectin extra domain-B) Ramucirumab VEGFR2 Rilotumumab HGFR Rituximab CD20 Robatumumab IGF-1R Sacituzumab govitecan Trop-2 (tumor-associated calcium signal transducer 2/EGP-1) Samalizumab CD200 (OX-2 membrane glycoprotein) Satumomab pendetide TAG-72 Seribantumab ERBB3 Seribantumab HER3 (ERBB3) SGN-CDA CD19 SGN-CDA CD33 Sibrotuzumab FAP Siltuximab IL-6 Simtuzumab LOXL2 Sofituzumab vedotin CA 125 Solitomab EpCAM Sonepcizumab S1P (sphingosine-1-phosphate) Tacatuzumab tetraxetan AFP (alpha-fetoprotein) Taplitumomab paptox CD19 Tarextumab Notch receptor Tenatumomab TN-C (tenascin C) Teprotumumab CD221 Tetulomab CD37 TGN CD28 Tigatuzumab TRAIL-R2 (CD262) Lebrikizumab IL-13 Tocilizumab IL-6R Tositumomab CD20 Tovetumab CD140a Tovetumab PDGFRα Trastuzumab HER2 (ERBB2/neu) Trastuzumab emtansine HER2 (ERBB2/neu) TRBS GD2 Tucotuzumab celmoleukin EpCAM ublituximab CD20 Ublituximab MS4A1 Ulocuplumab CXCR4 Vandortuzumab vedotin STEAP1 Vantictumab FZD7 Vanucizumab Ang-2 (angiopoietin 2) × VEGF-A Veltuzumab CD20 Vesencumab NRP1 Volociximab integrin α5β1 Votumumab CTAA16.88 Zalutumumab EGFR (HER1/ERBB1) Zanolimumab CD4 Zatuximab HER1 (EGFR/ERBB1)

Preferably, the neutralizing antibody is chosen from the list of anti-IL-10 and anti-TGFbeta.

In a particularly preferred embodiment, the at least one RNA of the RNA containing composition is an immunostimulating RNA, preferably according to SEQ ID NOs. 5, 394, or 10072 and is combined with at least one antibody, preferably anti-CD73 antibody or at least one decoy receptor as defined above, or a fragment or variant thereof.

Furthermore, the at least one antibody may preferably chosen from anti-CD73 antibodies or fragments or variants thereof.

In a further particularly preferred embodiment the at least one antibody is chosen from an antibody directed against CCR5/CD195 or from an antibody directed against its ligand CCL5/RANTES or fragments or variants thereof.

In a particularly preferred embodiment the decoy receptor is a soluble CCR5 (chemokine receptor type 5, also known as CD195).

In a particularly preferred embodiment the dominant negative receptor is dominant negative CCR5 (chemokine receptor type 5, also known as CD195).

13. Inhibitors of Myeloid Derived Suppressor Cells (MDSCs)

In this context protein-based inhibitors of myeloid derived suppressor cells (MDSCs), or fragments and variants thereof as disclosed above in the context of “coding RNA” may be used as additional pharmaceutically active component. Alternatively, nucleic acids encoding these proteins or peptides or fragments or variants thereof may be used as additional pharmaceutically active component.

It is particularly preferred to use anti IL-17 antibodies and IL-12 as inhibitors of MDSCs.

In the context of the invention, MDSC inhibition can be achieved by direct deactivation of MDSCs (e.g., chemical NO inhibitors (PDE-5 inhibitors, NO-aspirins, L-NAME), Arginase inhibitors (PDE-5 inhibitors, COX2 inhibitors, NOHA, L-NAME), ROS inhibitors (synthetic Triterpenoids)), by blocking differentiation of MDSCs into mature cells (e.g., ATRA, Vitamin A, Vitamin D3, CpG), by blocking the cell development of MDSCs (e.g. bisphosphorates (zolodronic acid), modulators of cell signaling (JAK2/STAT3 inhibitors, Multi-Kinase inhibitors, VEGF inhibitors)), or by depletion of MDSCs (e.g., cytotoxic agents (gemcitabine, cisplatin, paclitaxel, 5-fluorouracil) or HSP 90 inhibitors (17-DMAG)). Therefore these compounds may also be used as additional pharmaceutically active compound.

In a particularly preferred embodiment, the at least one RNA of the RNA containing composition is an immunostimulating RNA, preferably according to SEQ ID NOs. 5, 394, or 10072 and is combined with at least one inhibitor of MDSCs as defined above, or a fragment or variant thereof.

14. IDO Pathway Inhibitors

In this context protein-based IDO pathway inhibitors, or fragments and variants thereof as disclosed above in the context of “coding RNA” may be used as additional pharmaceutically active component. Alternatively, nucleic acids encoding these proteins or peptides or fragments or variants thereof may be used as additional pharmaceutically active component.

Chemical IDO Pathway Inhibitor:

In a further preferred embodiment the additional pharmaceutically active component may be selected from an IDO pathway inhibitor, which is preferably selected from small molecule inhibitor. Preferably the IDO pathway inhibitor is chosen from the following list: Indoximod (the D isomer of 1-methyl-tryptophan) and NLG919).

In a particularly preferred embodiment, the at least one RNA of the RNA containing composition is an immunostimulating RNA, preferably according to SEQ ID NOs. 5, 394, or 10072 and is combined with at least one IDO pathway inhibitor as defined above, or a fragment or variant thereof.

15. Proteins or Peptides that Bind Inhibitors of Apoptosis

Apoptosis is a tightly regulated cellular process and faulty regulation of apoptosis is a hallmark of human cancers. Targeting key apoptosis regulators with the goal to restore apoptosis in tumor cells has been pursued as a new cancer therapeutic strategy. XIAP, cIAP1, and cIAP2, members of inhibitor of apoptosis (IAP) proteins, are critical regulators of cell death and survival and are attractive targets for new cancer therapy. The SMAC/DIABLO protein is an endogenous antagonist of XIAP, cIAP1, and cIAP2. In the last decade, intense research efforts have resulted in the design and development of several small-molecule SMAC mimetics now in clinical trials for cancer treatment

In a further preferred embodiment, the inventive composition comprises at least one molecule that binds inhibitors of apoptosis proteins (IAPB) and thus sensitize cancer cells to apoptotic death.

Therefore it is particularly preferred that the inventive RNA containing composition comprises at least one molecule that binds inhibitors of apoptosis, such as SMAC mimetics.

Particularly preferred proteins or peptides that bind IAPB according to the present invention comprise Omi/HtrA2, Smac, Smac derived peptides, Smac/DIABLO, and XAF1 (XIAP-associated factor 1) and fragments or variants thereof.

In this context proteins or peptides that bind inhibitors of apoptosis, or fragments and variants thereof as disclosed above in the context of “coding RNA” may be used as additional pharmaceutically active component. Alternatively, nucleic acids encoding these proteins or peptides or fragments or variants thereof may be used as additional pharmaceutically active component.

Therefore it is particularly preferred that the additional pharmaceutically active component is selected from proteins or peptides that bind inhibitors of apoptosis, such as SMAC mimetics. Furthermore it is particularly preferred that such SMAC mimetics used as additional pharmaceutically active component are small molecules inhibiting inhibitors of apoptosis.

In a particularly preferred embodiment, the at least one RNA of the RNA containing composition is an immunostimulating RNA, preferably according to SEQ ID NOs. 5, 394, or 10072 and is combined with at least one proteins or peptides that bind inhibitors of apoptosis as defined above, or a fragment or variant thereof.

16. Anti-Bacterial Agent:

According to the present invention, the at least one additional pharmaceutically active component which may be contained in the inventive composition, and/or which may be co-administered, may be an anti-bacterial agent. In this context, any anti-bacterial agents known to one of skill in the art may be used in combination with the components of the inventive composition as defined herein. Non-limiting examples of anti-bacterial agents include Amikacin, Amoxicillin, Amoxicillin-clavulanic acid, Amphothericin-B, Ampicillin, Ampicllin-sulbactam, Apramycin, Azithromycin, Aztreonam, Bacitracin, Benzylpenicillin, Caspofungin, Cefaclor, Cefadroxil, Cefalexin, Cefalothin, Cefazolin, Cefdinir, Cefepime, Cefixime, Cefmenoxime, Cefoperazone, Cefoperazone-sulbactam, Cefotaxime, Cefoxitin, Cefbirome, Cefpodoxime, Cefpodoxime-clavulanic acid, Cefpodoxime-sulbactam, Cefbrozil, Cefquinome, Ceftazidime, Ceftibutin, Ceftiofur, Ceftobiprole, Ceftriaxon, Cefuroxime, Chloramphenicole, Florfenicole, Ciprofloxacin, Clarithromycin, Clinafloxacin, Clindamycin, Cloxacillin, Colistin, Cotrimoxazol (Trimthoprim/sulphamethoxazole), Dalbavancin, Dalfopristin/Quinopristin, Daptomycin, Dibekacin, Dicloxacillin, Doripenem, Doxycycline, Enrofloxacin, Ertapenem, Erythromycin, Flucloxacillin, Fluconazol, Flucytosin, Fosfomycin, Fusidic acid, Garenoxacin, Gatifloxacin, Gemifloxacin, Gentamicin, Imipenem, Itraconazole, Kanamycin, Ketoconazole, Levofloxacin, Lincomycin, Linezolid, Loracarbef, Mecillnam (amdinocillin), Meropenem, Metronidazole, Meziocillin, Mezlocillin-sulbactam, Minocycline, Moxifloxacin, Mupirocin, Nalidixic acid, Neomycin, Netilmicin, Nitrofurantoin, Norfloxacin, Ofloxacin, Oxacillin, Pefloxacin, Penicillin V, Piperacillin, Piperacillin-sulbactam, Piperacillin-tazobactam, Rifampicin, Roxythromycin, Sparfloxacin, Spectinomycin, Spiramycin, Streptomycin, Sulbactam, Sulfamethoxazole, Teicoplanin, Telavancin, Telithromycin, Temocillin, Tetracyklin, Ticarcillin, Ticarcillin-clavulanic acid, Tigecycline, Tobramycin, Trimethoprim, Trovafloxacin, Tylosin, Vancomycin, Virginiamycin, and Voriconazole.

17. Anti-Viral Agents:

According to the present invention, the at least one additional pharmaceutically active component/compound, which may be contained in the inventive composition, and/or which may be co-administered, may be an anti-viral agents, preferably, but are not limited to, nucleoside analogs (e.g., zidovudine, acyclovir, gangcyclovir, vidarabine, idoxuridine, trifluridine, and ribavirin), foscarnet, amantadine, peramivir, rimantadine, saquinavir, indinavir, ritonavir, alpha-interferons and other interferons, AZT, t-705, zanamivir (Relenza®), and oseltamivir)(Tamiflu®). Other anti-viral agents include influenza virus vaccines, e.g., Fluarix® (Glaxo SmithKline), FluMist® (MedImmune Vaccines), Fluvirin® (Chiron Corporation), Flulaval® (GlaxoSmithKline), Afluria® (CSL Biotherapies Inc.), Agriflu® (Novartis) or Fluzone® (Aventis Pasteur).

18. Drugs:

In some embodiments, the additional pharmaceutically active component/compound may include at least one drug. The term “drug” is intended to include any substance that, when introduced or absorbed into the body of a living organism, alters normal bodily or cellular function. Some non-limiting examples of suitable drugs, including combinations and alternative forms of the drugs such as alternative salt forms, free acid form, free base forms, pro-drugs and hydrates, include: analgesics/antipyretics (e.g., aspirin, acetaminophen, ibuprofen, naproxen sodium, buprenorphine, propoxyphene hydrochloride, propoxyphene napsylate, meperidine hydrochloride, hydromorphone hydrochloride, morphine, oxycodone, codeine, dihydrocodeine bitartrate, pentazocine, hydrocodone bitartrate, levorphanol, diflunisal, trolamine salicylate, nalbuphine hydrochloride, mefenamic acid, butorphanol, choline salicylate, butalbital, phenyltoloxamine citrate, diphenhydramine citrate, methotrimeprazine, cinnamedrine hydrochloride, and meprobamate); antiasthmatics (e.g., ketotifen and traxanox); antibiotics (e.g., neomycin, streptomycin, chloramphenicol, cephalosporin, ampicillin, penicillin, tetracycline, and ciprofloxacin); antidepressants (e.g., nefopam, oxypertine, doxepin, amoxapine, trazodone, amitriptyline, maprotiline, phenelzine, desipramine, nortriptyline, tranylcypromine, fluoxetine, imipramine, imipramine pamoate, isocarboxazid, trimipramine, and protriptyline); antidiabetics (e.g., biguanides and sulfonylurea derivatives); antifungal agents (e.g., griseofulvin, ketoconazole, itraconazole, amphotericin B, nystatin, and candicidin); antihypertensive agents (e.g., propanolol, propafenone, oxyprenolol, nifedipine, reserpine, trimethaphan, phenoxybenzamine, pargyline hydrochloride, deserpidine, diazoxide, guanethidine monosulfate, minoxidil, rescinnamine, sodium nitroprusside, rauwolfia serpentine, alseroxylon, and phentolamine); anti-inflammatories (e.g., (non-steroidal) indomethacin, ketoprofen, flurbiprofen, naproxen, ibuprofen, ramifenazone, piroxicam, (steroidal) cortisone, dexamethasone, fluazacort, deflazacort, celecoxib, rofecoxib, hydrocortisone, prednisolone, and prednisone); antineoplastics (e.g., cyclophosphamide, actinomycin, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, mitomycin, methotrexate, fluorouracil, gemcitabine, carboplatin, carmustine (BCNU), methyl-CCNU, cisplatin, etoposide, camptothecin and derivatives thereof, phenesterine, paclitaxel and derivatives thereof, docetaxel and derivatives thereof, vinblastine, vincristine, goserelin, leuprolide, tamoxifen, interferon alfa, retinoic acid (ATRA), nitrogen mustard alkylating agents, and piposulfan); antianxiety agents (e.g., lorazepam, buspirone, prazepam, chlordiazepoxide, oxazepam, clorazepate dipotassium, diazepam, hydroxyzine pamoate, hydroxyzine hydrochloride, alprazolam, droperidol, halazepam, chlormezanone, and dantrolene); immunosuppressive agents (e.g., cyclosporine, azathioprine, mizoribine, and FK506 (tacrolimus)); antimigraine agents (e.g., ergotamine, propanolol, isometheptene mucate, and dichloralphenazone); sedatives/hypnotics (e.g., barbiturates such as pentobarbital, pentobarbital, and secobarbital; and benzodiazapines such as flurazepam hydrochloride, triazolam, and midazolam); antianginal agents (e.g., beta-adrenergic blockers; calcium channel blockers such as nifedipine, and diltiazem; and nitrates such as nitroglycerin, isosorbide dinitrate, pentearythritol tetranitrate, and erythrityl tetranitrate); antipsychotic agents (e.g., haloperidol, loxapine succinate, loxapine hydrochloride, thioridazine, thioridazine hydrochloride, thiothixene, fluphenazine, fluphenazine decanoate, fluphenazine enanthate, trifluoperazine, chlorpromazine, perphenazine, lithium citrate, and prochlorperazine); antimanic agents (e.g., lithium carbonate); antiarrhythmics (e.g., bretylium tosylate, esmolol, verapamil, amiodarone, encamide, digoxin, digitoxin, mexiletine, disopyramide phosphate, procainamide, quinidine sulfate, quinidine gluconate, quinidine polygalacturonate, flecamide acetate, tocamide, and lidocaine); antiarthritic agents (e.g., phenylbutazone, sulindac, penicillanine, salsalate, piroxicam, azathioprine, indomethacin, meclofenamate, gold sodium thiomalate, ketoprofen, auranofin, aurothioglucose, and tolmetin sodium); antigout agents (e.g., colchicine, and allopurinol); anticoagulants (e.g., heparin, heparin sodium, and warfarin sodium); thrombolytic agents (e.g., urokinase, streptokinase, and alteplase); antifibrinolytic agents (e.g., aminocaproic acid); hemorheologic agents (e.g., pentoxifylline); antiplatelet agents (e.g., aspirin); anticonvulsants (e.g., valproic acid, divalproex sodium, phenyloin, phenyloin sodium, clonazepam, primidone, phenobarbital, carbamazepine, amobarbital sodium, methsuximide, metharbital, mephobarbital, mephenyloin, phensuximide, paramethadione, ethotoin, phenacemide, secobarbital sodium, clorazepate dipotassium, and trimethadione); antiparkinson agents (e.g., ethosuximide); antihistamines/antipruritics (e.g., hydroxyzine, diphenhydramine, chlorpheniramine, brompheniramine maleate, cyproheptadine hydrochloride, terfenadine, clemastine fumarate, triprolidine, carbinoxamine, diphenylpyraline, phenindamine, azatadine, tripelennamine, dexchlorpheniramine maleate, methdilazine, and); agents useful for calcium regulation (e.g., calcitonin, and parathyroid hormone); antibacterial agents (e.g., amikacin sulfate, aztreonam, chloramphenicol, chloramphenicol palmitate, ciprofloxacin, clindamycin, clindamycin palmitate, clindamycin phosphate, metronidazole, metronidazole hydrochloride, gentamicin sulfate, lincomycin hydrochloride, tobramycin sulfate, vancomycin hydrochloride, polymyxin B sulfate, colistimethate sodium, and colistin sulfate); antiviral agents (e.g., interferon alpha, beta or gamma, zidovudine, amantadine hydrochloride, ribavirin, and acyclovir); antimicrobials (e.g., cephalosporins such as cefazolin sodium, cephradine, cefaclor, cephapirin sodium, ceftizoxime sodium, cefoperazone sodium, cefotetan disodium, cefuroxime axetil, cefotaxime sodium, cefadroxil monohydrate, cephalexin, cephalothin sodium, cephalexin hydrochloride monohydrate, cefamandole nafate, cefoxitin sodium, cefonicid sodium, ceforanide, ceftriaxone sodium, ceftazidime, cefadroxil, cephradine, and cefuroxime sodium; penicillins such as ampicillin, amoxicillin, penicillin G benzathine, cyclacillin, ampicillin sodium, penicillin G potassium, penicillin V potassium, piperacillin sodium, oxacillin sodium, bacampicillin hydrochloride, cloxacillin sodium, ticarcillin disodium, azlocillin sodium, carbenicillin indanyl sodium, penicillin G procaine, methicillin sodium, and nafcillin sodium; macrolides such as, azithromycin, clarithromycin, and erythromycins such as erythromycin ethylsuccinate, erythromycin, erythromycin estolate, erythromycin lactobionate, erythromycin stearate, and erythromycin ethylsuccinate; and tetracyclines such as tetracycline hydrochloride, doxycycline hyclate, and minocycline hydrochloride); anti-infectives (e.g., GM-CSF); bronchodilators (e.g., sympathomimetics such as epinephrine hydrochloride, metaproterenol sulfate, terbutaline sulfate, isoetharine, isoetharine mesylate, isoetharine hydrochloride, albuterol sulfate, albuterol, bitolterolmesylate, isoproterenol hydrochloride, terbutaline sulfate, epinephrine bitartrate, metaproterenol sulfate, epinephrine, and epinephrine bitartrate; anticholinergic agents such as ipratropium bromide; xanthines such as aminophylline, dyphylline, metaproterenol sulfate, and theophylline; mast cell stabilizers such as cromolyn sodium; inhalant corticosteroids such as beclomethasone dipropionate (BDP), and beclomethasone dipropionate monohydrate; salbutamol; ipratropium bromide; budesonide; salmeterol; xinafoate; triamcinolone; nedocromil sodium; flunisolide; fluticasone propionate; steroidal compounds and hormones (e.g., androgens such as danazol, testosterone cypionate, fluoxymesterone, ethyltestosterone, testosterone enathate, methyltestosterone; estrogens such as estradiol, estropipate, and conjugated estrogens; progestins such as methoxyprogesterone acetate, and norethindrone acetate; corticosteroids such as triamcinolone, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, dexamethasone acetate, prednisone, methylprednisolone acetate suspension, triamcinolone acetonide, methylprednisolone, prednisolone sodium phosphate, methylprednisolone sodium succinate, hydrocortisone sodium succinate, triamcinolone hexacetonide, hydrocortisone, hydrocortisone cypionate, prednisolone, fludrocortisone acetate, paramethasone acetate, prednisolone tebutate, prednisolone acetate, prednisolone sodium phosphate, and thyroid hormones such as levothyroxine sodium); hypoglycemic agents (e.g., human insulin, purified beef insulin, purified pork insulin, glyburide, metformin, chlorpropamide, glipizide, tolbutamide, and tolazamide); hypolipidemic agents (e.g., clofibrate, dextrothyroxine sodium, probucol, pravastitin, atorvastatin, lovastatin, and niacin); proteins (e.g., DNase, alginase, superoxide dismutase, and lipase); nucleic acids (e.g., anti-sense nucleic acids); agents useful for erythropoiesis stimulation (e.g., erythropoietin); antiulcer/antireflux agents (e.g., famotidine, cimetidine, and ranitidine hydrochloride); antinauseants/antiemetics (e.g., meclizine hydrochloride, nabilone, prochlorperazine, dimenhydrinate, promethazine hydrochloride, thiethylperazine, and scopolamine); as well as other drugs useful in the compositions and methods described herein include mitotane, halonitrosoureas, anthrocyclines, ellipticine, ceftriaxone, ketoconazole, ceftazidime, oxaprozin, valacyclovir, urofollitropin, famciclovir, flutamide, enalapril, itraconazole, buspirone, gabapentin, fosinopril, tramadol, acarbose, lorazepam, follitropin, omeprazole, fluoxetine, lisinopril, tramadol, levofloxacin, zafirlukast, interferon, growth hormone, interleukin, erythropoietin, granulocyte stimulating factor, nizatidine, bupropion, perindopril, erbumine, adenosine, alendronate, alprostadil, benazepril, betaxolol, bleomycin sulfate, dexfenfluramine, diltiazem, fentanyl, flecamide, gemcitabine, glatiramer acetate, granisetron, lamivudine, mangafodipir trisodium, mesalamine, metoprolol fumarate, metronidazole, miglitol, moexipril, monteleukast, octreotide acetate, olopatadine, paricalcitol, somatropin, sumatriptan succinate, tacrine, verapamil, nabumetone, trovafloxacin, dolasetron, zidovudine, finasteride, tobramycin, isradipine, tolcapone, enoxaparin, fluconazole, lansoprazole, terbinafine, pamidronate, didanosine, diclofenac, cisapride, venlafaxine, troglitazone, fluvastatin, losartan, imiglucerase, donepezil, olanzapine, valsartan, fexofenadine, calcitonin, and ipratropium bromide. In some embodiments, the drug may be water soluble. In some embodiments, the drug may not be water soluble

19. Combination with Standard Therapy

According to the present invention, the at least one additional pharmaceutically active component/compound which may be contained in the inventive composition, and/or which may be co-administered, may be selected from any standard therapy used for the treatment of the particular tumor or cancer disease, e.g any chemotherapy, checkpoint modulator, kinase inhibitor etc.

Adjuvants and Further Components:

According to the present invention, the at least one additional pharmaceutically active component/compound which may be contained in the inventive composition, and/or which may be co-administered may be an adjuvant. According to a specific embodiment, the inventive composition may comprise an adjuvant. In this context, an adjuvant may be understood as any compound, which is suitable to initiate or increase an immune response of the innate immune system, i.e. a non-specific immune response. With other words, when administered, the inventive composition preferably elicits an innate immune response due to the adjuvant, optionally contained therein. Preferably, such an adjuvant may be selected from an adjuvant known to a skilled person and suitable for the present case, i.e. supporting the induction of an innate immune response in a mammal.

Particularly preferred as adjuvants suitable for depot and delivery are cationic or polycationic compounds as defined above for the RNA of the inventive composition as vehicle, transfection or complexation agent.

Furthermore, the inventive composition may comprise one or more additional adjuvants which are suitable to initiate or increase an immune response of the innate immune system, i.e. a non-specific immune response, particularly by binding to pathogen-associated molecular patterns (PAMPs). With other words, when administered, the pharmaceutical composition preferably elicits an innate immune response due to the adjuvant, optionally contained therein. Preferably, such an adjuvant may be selected from an adjuvant known to a skilled person and suitable for the present case, i.e. supporting the induction of an innate immune response in a mammal.

Also such an adjuvant may be selected from any adjuvant known to a skilled person and suitable for the present case, i.e. supporting the induction of an innate immune response in a mammal and/or suitable for depot and delivery of the components of the inventive composition. Preferred as adjuvants suitable for depot and delivery are cationic or polycationic compounds as defined above. Likewise, the adjuvant may be selected from the group consisting of, e.g., cationic or polycationic compounds as defined above, from chitosan, TDM, MDP, muramyl dipeptide, pluronics, alum solution, aluminium hydroxide, ADJUMERTM (polyphosphazene); aluminium phosphate gel; glucans from algae; algammulin; aluminium hydroxide gel (alum); highly protein-adsorbing aluminium hydroxide gel; low viscosity aluminium hydroxide gel; AF or SPT (emulsion of squalane (5%), Tween 80 (0.2%), Pluronic L121 (1.25%), phosphate-buffered saline, pH 7.4); AVRIDINETM (propanediamine); BAY R1005TM ((N-(2-deoxy-2-L-leucylaminob-D-glucopyranosyl)-N-octadecyl-dodecanoyl-amide hydroacetate); CALCITRIOLTM (1-alpha,25-dihydroxy-vitamin D3); calcium phosphate gel; CAPTM (calcium phosphate nanoparticles); cholera holotoxin, cholera-toxin-A1-protein-A-D-fragment fusion protein, sub-unit B of the cholera toxin; CRL 1005 (block copolymer P1205); cytokine-containing liposomes; DDA (dimethyldioctadecylammonium bromide); DHEA (dehydroepiandrosterone); DMPC (dimyristoylphosphatidylcholine); DMPG (dimyristoylphosphatidylglycerol); DOC/alum complex (deoxycholic acid sodium salt); Freund's complete adjuvant; Freund's incomplete adjuvant; gamma inulin; Gerbu adjuvant (mixture of: i)N-acetylglucosaminyl-(P1-4)-N-acetylmuramyl-L-alanyl-D35 glutamine (GMDP), ii) dimethyldioctadecylammonium chloride (DDA), iii) zinc-L-proline salt complex (ZnPro-8); GM-CSF); GMDP (N-acetylglucosaminyl-(b1-4)-N-acetylmuramyl-L47 alanyl-D-isoglutamine); imiquimod (1-(2-methypropyl)-1H-imidazo[4,5-c]quinoline-4-amine); ImmTherTM (N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glycerol dipalmitate); DRVs (immunoliposomes prepared from dehydration-rehydration vesicles); interferongamma; interleukin-1beta; interleukin-2; interleukin-7; interleukin-12; ISCOMSTM; ISCOPREP 7.0.3. TM; liposomes; LOXORIBINETM (7-allyl-8-oxoguanosine); LT 5 oral adjuvant (E. coli labile enterotoxin-protoxin); microspheres and microparticles of any composition; MF59TM; (squalenewater emulsion); MONTANIDE ISA 51TM (purified incomplete Freund's adjuvant); MONTANIDE ISA 720TM (metabolisable oil adjuvant); MPLTM (3-Q-desacyl-4′-monophosphoryl lipid A); MTP-PE and MTP-PE liposomes ((N-acetyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy))-ethylamide, monosodium salt); MURAMETIDETM (Nac-Mur-L-Ala-D-Gln-OCH3); MURAPALMITINETM and DMURAPALMITINETM (Nac-Mur-L-Thr-D-isoGln-sn-glyceroldipalmitoyl); NAGO (neuraminidase-galactose oxidase); nanospheres or nanoparticles of any composition; NISVs (non-ionic surfactant vesicles); PLEURANTM (β-glucan); PLGA, PGA and PLA (homo- and co-polymers of lactic acid and glycolic acid; microspheres/nanospheres); PLURONIC L121TM; PMMA (polymethylmethacrylate); PODDSTM (proteinoid microspheres); polyethylene carbamate derivatives; poly-rA: poly-rU (polyadenylic acid-polyuridylic acid complex); polysorbate 80 (Tween 80); protein cochleates (Avanti Polar Lipids, Inc., Alabaster, Ala.); STIMULONTM (QS-21); Quil-A (Quil-A saponin); S-28463 (4-amino-otec-dimethyl-2-ethoxymethyl-1H-imidazo[4,5-c]quinoline-1-ethanol); SAF-1TM (“Syntex adjuvant formulation”); Sendai proteoliposomes and Sendai containing lipid matrices; Span-85 (sorbitan trioleate); Specol (emulsion of Marcol 52, Span 85 and Tween 85); squalene or Robane® (2,6,10,15,19,23-hexamethyltetracosan and 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane); stearyltyrosine (octadecyltyrosine hydrochloride); Theramid® (N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Aladipalmitoxypropylamide); Theronyl-MDP (TermurtideTM or [thr 1]-MDP; N-acetylmuramyl-Lthreonyl-D-isoglutamine); Ty particles (Ty-VLPs or virus-like particles); Walter-Reed liposomes (liposomes containing lipid A adsorbed on aluminium hydroxide), and lipopeptides, including Pam3Cys, in particular aluminium salts, such as Adju-phos, Alhydrogel, Rehydragel; emulsions, including CFA, SAF, IFA, MF59, Provax, TiterMax, Montanide, Vaxfectin; copolymers, including Optivax (CRL1005), L121, Poloaxmer4010), etc.; liposomes, including Stealth, cochleates, including BIORAL; plant derived adjuvants, including QS21, Quil A, Iscomatrix, ISCOM; adjuvants suitable for costimulation including Tomatine, biopolymers, including PLG, PMM, Inulin, microbe derived adjuvants, including Romurtide, DETOX, MPL, CWS, Mannose, CpG nucleic acid sequences, CpG7909, ligands of human TLR 1-10, ligands of murine TLR 1-13, ISS-1018, 35 IC31, Imidazoquinolines, Ampligen, Ribi529, IMOxine, IRIVs, VLPs, cholera toxin, heat-labile toxin, Pam3Cys, Flagellin, GPI anchor, LNFPIII/Lewis X, antimicrobial peptides, UC-1V150, RSV fusion protein, cdiGMP; and adjuvants suitable as antagonists including CGRP neuropeptide.

Particularly preferred, an adjuvant may be selected from adjuvants, which support induction of a Th1-immune response or maturation of naïve T-cells, such as GM-CSF, IL-12, IFNg, any RNA as defined herein, preferably an immunostimulatory RNA, CpG DNA, etc.

It is possible that the inventive composition contains besides the at least one RNA as described above further components which are selected from the group comprising: further antigens or further antigen-providing nucleic acids; a further immunotherapeutic agent; one or more auxiliary substances; or any further compound, which is known to be immunostimulating due to its binding affinity (as ligands) to human Toll-like receptors; and/or an adjuvant nucleic acid, preferably an immunostimulatory RNA (is RNA).

The inventive composition can additionally contain one or more auxiliary substances in order to increase its immunogenicity or immunostimulatory capacity, if desired. A synergistic action of the at least one RNA as defined herein and of an auxiliary substance, which may be optionally contained in the inventive composition, is preferably achieved thereby. Depending on the various types of auxiliary substances, various mechanisms can come into consideration in this respect. For example, compounds that permit the maturation of dendritic cells (DCs), for example lipopolysaccharides, TNF-alpha or CD40 ligand, form a first class of suitable auxiliary substances. In general, it is possible to use as auxiliary substance any agent that influences the immune system in the manner of a “danger signal” (LPS, GP96, etc.) or cytokines, such as GM-CFS, which allow an immune response to be enhanced and/or influenced in a targeted manner. Particularly preferred auxiliary substances are cytokines, such as monokines, lymphokines, interleukins or chemokines, that further promote the innate immune response, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IFN-alpha, IFN-beta, IFNgamma, GM-CSF, G-CSF, M-CSF, LT-beta or TNF-alpha, growth factors, such as hGH.

The inventive composition may contain any further compound, which is known to be immunostimulating due to its binding affinity (as ligands) to human Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, or due to its binding affinity (as ligands) to murine Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13.

Further additives which may be included in the inventive composition are emulsifiers, such as, for example, Tween; wetting agents, such as, for example, sodium lauryl sulfate; colouring agents; taste-imparting agents, pharmaceutical carriers; tablet-forming agents; stabilizers; antioxidants; preservatives.

Pharmaceutical Composition:

In a further aspect, the present invention also provides a pharmaceutical composition, comprising the RNA containing composition as defined herein and a pharmaceutically acceptable carrier and/or vehicle. Preferably the pharmaceutical composition is prepared for intratumoral application, preferably by injection into tumor tissue. Sterile injectable forms of the inventive pharmaceutical composition may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.

A pharmaceutically acceptable carrier typically includes the liquid or non-liquid basis of a composition comprising the components of the inventive composition. If the composition is provided in liquid form, the carrier will typically be pyrogen-free water; isotonic saline or buffered (aqueous) solutions, e.g. phosphate, citrate etc. buffered solutions. The injection buffer may be hypertonic, isotonic or hypotonic with reference to the specific reference medium, i.e. the buffer may have a higher, identical or lower salt content with reference to the specific reference medium, wherein preferably such concentrations of the afore mentioned salts may be used, which do not lead to damage of cells due to osmosis or other concentration effects. Reference media are e.g. liquids occurring in “in vivo” methods, such as blood, lymph, cytosolic liquids, or other body liquids, or e.g. liquids, which may be used as reference media in “in vitro” methods, such as common buffers or liquids. Such common buffers or liquids are known to a skilled person. Ringer-Lactate solution is particularly preferred as a liquid basis.

However, one or more compatible solid or liquid fillers or diluents or encapsulating compounds, which are suitable for administration to a patient to be treated, may be used as well for the pharmaceutical composition according to the invention. The term “compatible” as used here means that these constituents of the inventive pharmaceutical composition are capable of being mixed with the components of the inventive pharmaceutical composition in such a manner that no interaction occurs which would substantially reduce the pharmaceutical effectiveness of the pharmaceutical composition under typical use conditions.

Administration:

The inventive composition or the inventive pharmaceutical composition may be administered by conventional needle injection or needle-free jet injection into the tumor tissue. In a preferred embodiment the inventive composition or the inventive pharmaceutical composition is administered by jet injection. Jet injection refers to a needle-free injection method, wherein a fluid comprising the inventive composition and, optionally, further suitable excipients is forced through an orifice, thus generating an ultra-fine liquid stream of high pressure that is capable of penetrating mammalian skin. In principle, the liquid stream forms a hole in the skin, through which the liquid stream is pushed into the target tissue, namely the tumor tissue. According to the invention, jet injection may be used for intratumoral application of the inventive composition.

The inventive composition may be administered by conventional needle injection or needle-free jet injection adjacent to and/or in close proximity to the tumor tissue. In a preferred embodiment the inventive pharmaceutical composition is administered by jet injection adjacent to and/or in close proximity to the tumor tissue. Jet injection refers to a needle-free injection method, wherein a fluid comprising the inventive composition and, optionally, further suitable excipients is forced through an orifice, thus generating an ultra-fine liquid stream of high pressure that is capable of penetrating mammalian skin. In principle, the liquid stream forms a hole in the skin, through which the liquid stream is pushed into the target tissue, namely the tumor tissue. According to the invention, jet injection may be used for intratumoral application (adjacent to and/or in close proximity to the tumor tissue), particularly for injection of the inventive composition.

In other embodiments, the inventive composition or the inventive pharmaceutical composition may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intranodal, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, intracranial, transdermal, intradermal, intrapulmonal, intraperitoneal, intracardial, intraarterial, and sublingual injection or infusion techniques.

Further particularly preferred administration routes are intradermal and intramuscular injection.

Despite, the inventive pharmaceutical composition may comprise further components for facilitating administration and uptake of components of the pharmaceutical composition. Such further components may be an appropriate carrier or vehicle, additional adjuvants for supporting any immune response, antibacterial and/or antiviral agents.

A further component of the inventive pharmaceutical composition may be an immunotherapeutic agent that can be selected from immunoglobulins, preferably IgGs, monoclonal or polyclonal antibodies, polyclonal serum or sera, etc. Preferably, such a further immunotherapeutic agent may be provided as a peptide/protein or may be encoded by a nucleic acid, preferably by a DNA or an RNA, more preferably an mRNA.

The inventive pharmaceutical composition typically comprises a “safe and effective amount” of the components of the inventive pharmaceutical composition, particularly of the RNA molecule(s) as defined herein. As used herein, a “safe and effective amount” means an amount of the RNA molecule(s) as defined herein as such that is sufficient to significantly induce a positive modification of the tumor or cancer disease. At the same time, however, a “safe and effective amount” is small enough to avoid serious side-effects and to permit a sensible relationship between advantage and risk. The determination of these limits typically lies within the scope of sensible medical judgment.

The inventive pharmaceutical composition may be used for human and also for veterinary medical purposes, preferably for human medical purposes, as a pharmaceutical composition in general.

Vaccine:

According to another particularly preferred aspect, the inventive composition or the inventive pharmaceutical composition may be provided or used as a vaccine. Typically, such a vaccine is as defined above for pharmaceutical compositions. Additionally, such a vaccine typically contains the at least one RNA as defined herein or the inventive composition comprising a plurality of RNAs. Preferably, the at least one RNA encodes at least one tumor antigen or at least one immune activator as defined above. The inventive vaccine may also comprise a pharmaceutically acceptable carrier, adjuvant, and/or vehicle as defined herein for the inventive pharmaceutical composition. In the specific context of the inventive vaccine, the choice of a pharmaceutically acceptable carrier is determined in principle by the manner in which the inventive vaccine is administered. The inventive vaccine may be administered locally into tumor tissue.

The inventive vaccine can additionally contain one or more auxiliary substances in order to increase its immunogenicity or immunostimulatory capacity, if desired. Particularly preferred are adjuvants as auxiliary substances or additives as defined for the pharmaceutical composition.

Kit or Kit of Parts:

In a further aspect, the invention relates to a kit or kit of parts comprising the RNA containing composition as described above, or comprising the pharmaceutical composition as described above, or the components thereof and optionally technical instructions with information on the administration and dosage of the components.

Beside the components of the inventive RNA containing composition, the kit may additionally contain a pharmaceutically acceptable vehicle, an adjuvant and at least one further component e.g. an additional pharmaceutically active component/compound as defined herein, as well as means for administration and technical instructions. The components of the composition and possibly further components may be provided in lyophilized form. In a preferred embodiment, prior to use of the kit, the provided vehicle is than added to the lyophilized components in a predetermined amount as written e.g. in the provided technical instructions.

Medical Indication:

The present invention furthermore provides several applications and uses of the inventive RNA containing composition, or the pharmaceutical composition, or the vaccine, or the kit or kit of parts as defined herein. As a main aspect of the invention the composition or the pharmaceutical composition or the kit or kit of parts may be used as a medicament, namely for treatment of tumor or cancer diseases. In this context the treatment is preferably done by intratumoral application, especially by injection into tumor tissue. According to another aspect, the present invention is directed to the second medical use of the RNA containing composition or the pharmaceutical composition, or the vaccine, or the kit or kit of parts as described above, wherein these subject matters are used for preparation of a medicament particularly for intratumoral application (administration) for treatment of tumor or cancer diseases.

Preferably, diseases as mentioned herein are selected from tumor or cancer diseases which preferably include e.g. Acute lymphoblastic leukemia, Acute myeloid leukemia, Adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, Anal cancer, Appendix cancer, Astrocytoma, Basal cell carcinoma, Bile duct cancer, Bladder cancer, Bone cancer, Osteosarcoma/Malignant fibrous histiocytoma, Brainstem glioma, Brain tumor, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, Breast cancer, Bronchial adenomas/carcinoids, Burkitt lymphoma, childhood Carcinoid tumor, gastrointestinal Carcinoid tumor, Carcinoma of unknown primary, primary Central nervous system lymphoma, childhood Cerebellar astrocytoma, childhood Cerebral astrocytoma/Malignant glioma, Cervical cancer, Childhood cancers, Chronic lymphocytic leukemia, Chronic myelogenous leukemia, Chronic myeloproliferative disorders, Colon Cancer, Cutaneous T-cell lymphoma, Desmoplastic small round cell tumor, Endometrial cancer, Ependymoma, Esophageal cancer, Ewing's sarcoma in the Ewing family of tumors, Childhood Extracranial germ cell tumor, Extragonadal Germ cell tumor, Extrahepatic bile duct cancer, Intraocular melanoma, Retinoblastoma, Gallbladder cancer, Gastric (Stomach) cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal stromal tumor (GIST), extracranial, extragonadal, or ovarian Germ cell tumor, Gestational trophoblastic tumor, Glioma of the brain stem, Childhood Cerebral Astrocytoma, Childhood Visual Pathway and Hypothalamic Glioma, Gastric carcinoid, Hairy cell leukemia, Head and neck cancer, Heart cancer, Hepatocellular (liver) cancer, Hodgkin lymphoma, Hypopharyngeal cancer, childhood Hypothalamic and visual pathway glioma, Intraocular Melanoma, Islet Cell Carcinoma (Endocrine Pancreas), Kaposi sarcoma, Kidney cancer (renal cell cancer), Laryngeal Cancer, Leukemias, acute lymphoblastic Leukemia, acute myeloid Leukemia, chronic lymphocytic Leukemia, chronic myelogenous Leukemia, hairy cell Leukemia, Lip and Oral Cavity Cancer, Liposarcoma, Liver Cancer, Non-Small Cell Lung Cancer, Small Cell Lung Cancer, Lymphomas, AIDS-related Lymphoma, Burkitt Lymphoma, cutaneous T-Cell Lymphoma, Hodgkin Lymphoma, Non-Hodgkin Lymphomas, Primary Central Nervous System Lymphoma, Waldenström Macroglobulinemia, Malignant Fibrous Histiocytoma of Bone/Osteosarcoma, Childhood Medulloblastoma, Melanoma, Intraocular (Eye) Melanoma, Merkel Cell Carcinoma, Adult Malignant Mesothelioma, Childhood Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Mouth Cancer, Childhood Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma/Plasma Cell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes, Myelodysplastic/Myeloproliferative Diseases, Chronic Myelogenous Leukemia, Adult Acute Myeloid Leukemia, Childhood Acute Myeloid Leukemia, Multiple Myeloma (Cancer of the Bone-Marrow), Chronic Myeloproliferative Disorders, Nasal cavity and paranasal sinus cancer, Nasopharyngeal carcinoma, Neuroblastoma, Oral Cancer, Oropharyngeal cancer, Osteosarcoma/malignant fibrous histiocytoma of bone, Ovarian cancer, Ovarian epithelial cancer (Surface epithelial-stromal tumor), Ovarian germ cell tumor, Ovarian low malignant potential tumor, Pancreatic cancer, islet cell Pancreatic cancer, Paranasal sinus and nasal cavity cancer, Parathyroid cancer, Penile cancer, Pharyngeal cancer, Pheochromocytoma, Pineal astrocytoma, Pineal germinoma, childhood Pineoblastoma and supratentorial primitive neuroectodermal tumors, Pituitary adenoma, Plasma cell neoplasia/Multiple myeloma, Pleuropulmonary blastoma, Primary central nervous system lymphoma, Prostate cancer, Rectal cancer, Renal cell carcinoma (kidney cancer), Cancer of the Renal pelvis and ureter, Retinoblastoma, childhood Rhabdomyosarcoma, Salivary gland cancer, Sarcoma of the Ewing family of tumors, Kaposi Sarcoma, soft tissue Sarcoma, uterine Sarcoma, Sézary syndrome, Skin cancer (nonmelanoma), Skin cancer (melanoma), Merkel cell Skin carcinoma, Small intestine cancer, Squamous cell carcinoma, metastatic Squamous neck cancer with occult primary, childhood Supratentorial primitive neuroectodermal tumor, Testicular cancer, Throat cancer, childhood Thymoma, Thymoma and Thymic carcinoma, Thyroid cancer, childhood Thyroid cancer, Transitional cell cancer of the renal pelvis and ureter, gestational Trophoblastic tumor, Urethral cancer, endometrial Uterine cancer, Uterine sarcoma, Vaginal cancer, childhood Visual pathway and hypothalamic glioma, Vulvar cancer, Waldenström macroglobulinemia, and childhood Wilms tumor (kidney cancer).

Especially preferred examples of tumors or cancers that are suitable for intratumoral administration are prostate cancer, lung cancer, breast cancer, brain cancer, head and neck cancer, thyroid cancer, colon cancer, stomach cancer, liver cancer, pancreas cancer, ovary cancer, skin cancer, urinary bladder, uterus and cervix.

According to a specific embodiment, the medicament may be administered to the patient as a single dose or as several doses. In certain embodiments, the medicament may be administered to a patient as a single dose followed by a second dose later and optionally even a third, fourth (or more) dose subsequent thereto etc.

Preferably, the inventive composition is provided in an amount of at least 40 μg RNA per dose. More specifically, the amount of the mRNA comprised in a single dose is typically at least 200 μg, preferably from 200 μg to 1.000 μg, more preferably from 300 μg to 850 μg, even more preferably from 300 μg to 700 μg.

Treatment with Additional (Pharmaceutical) Compounds:

In a particularly preferred embodiment the subject receiving the inventive composition, or the pharmaceutical composition or vaccine may be a patient with cancer or tumor who receives or received standard treatments of cancer. Preferably, the patient has achieved partial response or stable disease after having received standard treatments.

The standard treatments of cancer include chemotherapy, radiation, chemoradiation and surgery dependent on the particular cancer or tumor type to be treated, wherein these treatments are applied individually or in combination.

In some embodiments the subject receiving the inventive composition, pharmaceutical composition or vaccine may be a patient with cancer or tumor, who received or receives chemotherapy (e.g. first-line or second-line chemotherapy), radiotherapy, chemoradiation (combination of chemotherapy and radiotherapy), kinase inhibitors, antibody therapy and/or checkpoint modulators (e.g. CTLA4 inhibitors, PD1 pathway inhibitors), or a patient, who has achieved partial response or stable disease after having received one or more of the treatments specified above.

In other embodiments the subject receiving the inventive composition, pharmaceutical composition or vaccine may be a patient with cancer or tumor, who received or receives an additional pharmaceutically active component/compound as defined above. Preferably, the subject is a patient, who has achieved partial response or stable disease after having received one or more of the treatments specified above.

According to a further aspect the invention refers to a method of treatment of tumor or cancer diseases, wherein the RNA containing composition as described above, or the pharmaceutical composition as described above, or the vaccine as described above, or the kit or kit of parts as described above is preferably applied intratumorally, especially by injection into tumor tissue. With respect to further features of the method for treatment it is referred to the description above.

Preferred Intratumoral Applications:

In this context it is particularly preferred that the intratumoral application of the RNA containing composition or the pharmaceutical composition or the vaccine as defined above is combined with the application of different agents/pharmaceutically active components/compounds. Particularly preferred are antibodies (e.g. check point modulators as e.g. anti-CTLA4, anti-OX40, anti-PD1 or anti-PD-L1) or ligands (e.g. CD40L).

In preferred embodiments the following combinations are particularly preferred:

-   -   RNAdjuvant (i.t.)+anti-CTLA4 as protein (i.p./i.v.)     -   RNAdjuvant (i.t.)+anti-CTLA4 as protein (i.t.)     -   RNAdjuvant (i.t.)+anti-PD1 as protein (i.p./i.v.)     -   RNAdjuvant (i.t.)+anti-PD1 as protein (i.t.)     -   RNAdjuvant (i.t.)+anti-PD-L1 as protein (i.p./i.v.)     -   RNAdjuvant (i.t.)+anti-PD-L1 as protein (i.t.)     -   RNAdjuvant (i.t.)+CD40L (i.t.) as protein or encoded by a         nucleic acid preferably an RNA, more preferably an mRNA     -   RNAdjuvant (i.t.)+mRNA encoding IL-12+mRNA encoding soluble PD-1         receptor+anti-CD73 (i.p./i.v.)     -   RNAdjuvant (i.t.)+mRNA encoding IL-12+mRNA encoding soluble PD-1         receptor+anti-CD137 (i.p./i.v.)     -   RNadjuvant (i.t.)

(i.t.=intratumoral, i.p.=intraperitoneal, i.v.=intravenous)

In the present invention, if not otherwise indicated, different features of alternatives and embodiments may be combined with each other, where suitable. Furthermore, the term “comprising” shall not be narrowly construed as being limited to “consisting of” only, if not specifically mentioned. Rather, in the context of the present invention, “consisting of” is an embodiment specifically contemplated by the inventors to fall under the scope of “comprising”, wherever “comprising” is used herein.

All publications, patents and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

The examples and figures shown in the following are merely illustrative and shall describe the present invention in a further way. These figures and examples shall not be construed to limit the present invention thereto.

SHORT DESCRIPTION OF THE FIGURES

FIG. 1: shows survival proportions of mice bearing E.G7-OVA tumors after intratumoral treatment with mRNA encoding IL-12 (IL-12 mRNA) or with recombinant IL-12 protein (rIL-12 protein). The experiment was performed as described in Example 1. Kaplan-Meier survival curves are presented.

FIG. 2: shows that intratumoral treatment of mice with a combination of IL-12 mRNA (R2763, SEQ ID NO: 1) and the polymeric carrier cargo complex (R2391, RNAdjuvant®, prepared as described in methods) led to a significantly decreased tumor volume compared to control groups. The experiment was performed as described in Example 2. FIG. 2 shows the mean tumor volume at day 21 after tumor challenge (the last day when all animals were alive). Statistical analysis was performed in GraphPad Prism version 5.04 using Mann Whitney test.

FIG. 3: shows survival proportions of mice bearing CT26 tumors after intratumoral treatment with a combination of IL-12 mRNA and the polymeric carrier cargo complex (“RNAdjuvant”) as described in Example 2 and in legend of FIG. 2. Kaplan-Meier survival curves are presented. Statistical analysis was performed in GraphPad Prism version 5.04 using Log-rank test.

FIG. 4: shows survival proportions of mice bearing CT26 tumors after intratumoral treatment with mRNA encoding the influenza nucleoprotein. The experiment was performed as described in Example 3. Kaplan-Meier survival curves are presented.

FIGS. 5A-B: Panel (A) shows an analysis of the median tumor growth of mice bearing CT26 tumors after intratumoral treatment with mRNA encoding IL-12, RNAdjuvant, and mRNA encoding soluble PD-1. Respective combinations of these compounds, including control groups, were tested as indicated in the figure. The experiment was performed as described in Example 4.

-   -   Panel (B) shows survival proportions of mice bearing CT26 tumors         after intratumoral treatment with mRNA encoding IL-12,         RNAdjuvant, and mRNA encoding soluble PD-1. Respective         combinations of these compounds, including control groups, were         tested as indicated in the figure. The experiment was performed         as described in Example 4. Kaplan-Meier survival curves are         presented.

FIG. 6: shows survival proportions of mice bearing CT26 tumors after intratumoral treatment with mRNA encoding IL-12, RNAdjuvant, mRNA encoding soluble PD-1 and intraperitoneal treatment of an anti-CD73 antibody. Respective combinations of these compounds, including control groups, were tested as indicated in the figure. The experiment was performed as described in Example 5. Kaplan-Meier survival curves are presented.

FIG. 7: shows survival proportions of mice bearing CT26 tumors after intratumoral treatment with mRNA encoding IL-12, RNAdjuvant, mRNA encoding soluble PD-1 and intraperitoneal treatment of an anti-CD173 antibody. Respective combinations of these compounds, including control groups, were tested as indicated in the figure. The experiment was performed as described in Example 6. Kaplan-Meier survival curves are presented.

FIG. 8: shows survival proportions of mice bearing CT26 tumors after intratumoral treatment with RNAdjuvant and intraperitoneal treatment of an anti-PD-1 antibody. Respective combinations of these compounds, including control groups, were tested as indicated in the figure. The experiment was performed as described in Example 7. Kaplan-Meier survival curves are presented.

FIG. 9: shows survival proportions of mice bearing CT26 tumors after intratumoral treatment with mRNA encoding IL-12, RNAdjuvant, mRNA encoding CD40L compared to intratumoral treatment with mRNA encoding IL-12 alone. Respective combinations of these compounds, including control groups, were tested as indicated in the figure. The experiment was performed as described in Example 8. Kaplan-Meier survival curves are presented.

FIG. 10: shows the mRNA sequence R3571 encoding murine CD40L (MmCD40L) according to SEQ ID NO. 10.073

EXAMPLES Methods: Preparation of the RNA

1. Preparation of DNA and RNA Constructs

For the present examples DNA sequences encoding the indicated RNAs (see Table 17) were prepared and used for subsequent RNA in vitro transcription reactions.

TABLE 17 RNA constructs RNA Description 5′-UTR 3′-UTR SEQ ID NO. R1328 Murine IL-12 encoding mRNA — Muag (3′-UTR of SEQ ID NO: (MmIL-12(GC))-sc-Flag) alpha globin)-A64- 1 C30 R491  mRNA encoding Photinus — Muag (3′-UTR of SEQ ID NO: pyralis luciferase (pPLuc (GC)) alpha globin)-A64- 2 (irrelevant mRNA) C30 R2763 Murine IL-12 encoding mRNA 5′-TOP-UTR albumin-3′-UTR- SEQ ID NO: (MmIL-12 (GC)) derived from A64-C30-histone 3 the ribosomal stem-loop protein 32 L R2244 Luciferase encoding mRNA 5′-TOP-UTR albumin-3′-UTR- SEQ ID NO: (PpLuc(GC)) derived from A64-C30-histone 4 the ribosomal stem-loop protein 32 L R2025 Non-coding SEQ ID NO: R2391 immunostimulatory RNA 5 (RNAdjuvant) (SEQ ID NO. 118 of WO2009095226) R2650 mRNA coding for the 5′-TOP-UTR albumin-3′-UTR- SEQ ID NO: R2651 influenza nucleoprotein derived from A64-C30-histone 6 (H1N1(PR8)-NP(GC)) the ribosomal stem-loop protein 32 L R3971 mRNA encoding solPD-1 5′-TOP-UTR albumin-3′-UTR- SEQ ID NO: derived from A64-C30-histone 389 the ribosomal stem-loop protein 32 L R3571 mRNA encoding murine 5′-TOP-UTR albumin-3′-UTR- SEQ ID NO: CD40L (MmCD40L) derived from A64-C30-histone 10.073 the ribosomal stem-loop protein 32 L

The constructs of MmIL-12(GC), Influenza NP (GC), soIPD-1 and PpLuc(GC)) were prepared by introducing a 5′-TOP-UTR derived from the ribosomal protein 32 L, modifying the wild type coding sequence by introducing a GC-optimized sequence for stabilization, followed by a stabilizing sequence derived from the albumin-3′-UTR, a stretch of 64 adenosines (poly(A)-sequence), a stretch of 30 cytosines (poly(C)-sequence), and a histone stem loop. Most DNA sequences were prepared by modifying the wild type encoding DNA sequences by introducing a GC-optimized sequence for stabilization, using an in silico algorithm that increase the GC content of the respective coding sequence compared to the wild type coding sequence (in Table 12 indicated as “GC”).

For the present example a DNA sequence encoding the non-coding immunostimulatory RNA (isRNA) R2025 was prepared and used for subsequent RNA in vitro transcription reactions.

2. RNA In Vitro Transcription

The respective DNA plasmids prepared according to section 1 above were transcribed in vitro using T7 polymerase. The RNA in vitro transcription reactions of the IL-12, the NP, PpLuc, CD40L and soluble PD-1 encoding constructs were performed in the presence of a CAP analog (m⁷GpppG). The isRNA R2025 was prepared without CAP analog. Subsequently, the RNA was purified using PureMessenger® (CureVac, Tübingen, Germany; WO2008/077592A1).

3. Preparation of the Polymeric Cargo Complex (“RNAdjuvant”)

The following cationic peptide as cationic component of the polymeric carrier was used (Cys-Arg₁₂-Cys or CR₁₂C) according to SEQ ID NO: 7.

For synthesis of the polymeric carrier cargo complexes an RNA molecule having the RNA sequence R2025 as defined in section 1 above was mixed with the cationic CR12C peptide component as defined above. The specified amount of the RNA was mixed with the respective cationic component in mass ratios as indicated below, thereby forming a complex. If polymerizing cationic components were used according to the present invention, polymerization of the cationic components took place simultaneously to complexation of the nucleic acid cargo. Afterwards, the resulting solution was adjusted with water to a final volume of 50 μl and incubated for 30 minutes at room temperature. Further details are described in WO2012013326.

The mass ratio of peptide:RNA was 1:3.7. The polymeric carrier cargo complex is formed by the disulfide-crosslinked cationic peptide CR12C as carrier and the immunostimulatory R2025 as nucleic acid cargo. This polymeric carrier cargo complex R2025/CR12C (designated R2391) was used as adjuvant in the following examples (referred to as “RNAdjuvant”)

4. Preparation of the Vaccine Formulation Coding for the Influenza Nucleoprotein (H1N1(PR8)-NP(GC)) (R2651)

The mRNA (R2650) was complexed with protamine by addition of protamine to the mRNA in the ratio (1:2) (w/w) (adjuvant component). After incubation for 10 min, the same amount of free mRNA used as antigen-providing mRNA was added. This vaccine formulation is termed herein R2651 (according to WO2010037539). The vaccine was administered in Ringer's Lactate solution.

5. Preparation of the RNA for Administration

The naked (that is, non-formulated) PpLuc mRNA (R2244, R491)), IL-12 mRNA (R2763, R1328), soluble PD-1 mRNA (R3971), CD40L mRNA (R3571) were administered in Ringer's Lactate (RiLa) solution. The co-formulation of naked mRNAs and the polymeric carrier cargo complex “RNAdjuvant” (R2391) were also administered in Ringer's Lactate (RiLa) after mixing of both components directly before injection.

Example 1: Intratumoral Application of mRNA Coding for IL-12

5 female C57BL/6 mice per treatment group were inoculated with 10⁶ cells E.G7-OVA cells 5 days before the first treatment. For each treatment group 5 established (about 100 mm³) subcutaneously implanted EG.7-OVA tumors were treated. Tumors were treated with 16 μg mRNA coding for MmIL-12 (MmIL-12(GC))-sc-Flag) (R1328) or 0.5 μg MmIL-12 protein on d 0, 2, 4, 21, 23 and 25 with 50 μg (1 μg/μl). As control mice were treated with an irrelevant mRNA (pPLuc) (R491).

Study day 0 is defined as the first day of treatment. Tumor growth was monitored frequently (every 2-3 days). Mice with a volume of >3 cm³ were killed.

Results of Example 1

FIG. 1 shows that the intratumoral treatment with the mRNA-encoded IL-12 (IL-12 mRNA) resulted in a significant increase in survival compared to the control group. Furthermore an increased survival could be observed compared to the intratumoral application of recombinant IL-12 protein (rIL-12 protein).

Example 2: Intratumoral Treatment with mRNA Encoding IL-12 in Combination with an Immunostimulating RNA (RNAdjuvant®)

The following table 18 summarizes the RNA constructs used for the example 2.

TABLE 18 RNA constructs for example 2 RNA Description FIG. SEQ ID NO. R2763 Murine IL-12 encoding mRNA 1 SEQ ID NO. 1 R2244 Luciferase encoding mRNA (PpLuc) 2 SEQ ID NO. 2 R2025 Non-coding immunostimulatory RNA 3 SEQ ID NO. 3

Balb/c mice (n=6 or 7, see table 14) were injected subcutaneously (s.c.) with 1×10⁶ CT26 cells (colon carcinoma cell line) per mouse (in a volume of 100 μl PBS) on the right flank on day 0 of the experiment. At day 9 after tumor challenge, mice were sorted according to the tumor size to obtain groups with a mean tumor volume of approximately 60 mm³. Intratumoral (i.t.) therapy started at day 9 and continued for additional 4 injections every 3-4 days. Mice were injected with a combination of mRNA-encoded IL-12 (25 μg of R2763)+RNAdjuvant® (25 μg of R2391) (group A). To control for local inflammation due to RNA application or the injection procedure, mice were injected with control mRNA coding for luciferase (PpLuc, R2244, group B) or buffer (RiLa, group C), respectively. Untreated mice served as additional control (group D).

Tumor growth was monitored by measuring the tumor size in three dimensions using a calliper. Tumor volume was calculated according to the following formula:

${{volume}\mspace{14mu}\left( {mm}^{3} \right)} = \frac{{length}\mspace{14mu}({mm}) \times \pi \times {{width}^{2}\left( {mm}^{2} \right)}}{6}$

On day 9, 11, 14, 17 and 21 of the experiment mice were injected intratumorally (i.t.) with RNA according to the table 19 below. The volume for intratumoral injection was 50 μl.

TABLE 19 Animal groups Strain Number Dose per Route, Group sex of mice RNA mouse volume A BALB/c 7  R2763, 25 μg i.t., Female R2391 of each 50 μl RNA B BALB/c 7 R2244 50 μg i.t., Female 50 μl C BALB/c 6 RiLa — i.t., Female 50 μl D BALB/c 6 — — — Female

Results of Example 2

FIG. 2 shows that the intratumoral treatment with the combination of mRNA-encoded IL-12 (R2763) and RNAdjuvant® (R2391) resulted in a statistically significant decrease in tumor volume at day 21 after tumor challenge compared to all control groups.

FIG. 3 shows that the intratumoral treatment with the combination of mRNA-encoded IL-12 (R2763) and RNAdjuvant® (R2391) resulted in a statistically significant increase in survival compared to all three control groups (group A vs. group B*p=0.0104, group A vs. group C**p=0.0035, group A vs. Group D*p==0.0263).

Example 3: Vaccination of Mice with mRNA Encoding the Influenza Nucleoprotein (NP) and Subsequent Intratumoral Treatment with NP-Encoding mRNA

The objective of this experiment was to test whether a pre-existing immune response can be harnessed against an established tumor. To this end, mice were first vaccinated with RNActive (vaccine formulation complexed with protamine) encoding the influenza nucleoprotein (NP) (R2651) which induces a high level of anti-NP CD8+ T cell responses, then challenged with CT26 tumor cells followed by intratumoral treatment with naked RNA encoding NP (R2650).

27 Balb/c mice were vaccinated intradermally (i.d.) with 40 μg of H1N1(PR8)-NP(GC) RNActive (R2651) (2×50 up or Ringer-Lactate buffer (RiLa) as control on day 0, day 7 and day 16 of the experiment. On day 14 all mice were challenged subcutaneously (s.c.) with 1×10⁶ CT26 cells per mouse (in a volume of 100 μl PBS) on the right flank. On day 22, mice were assigned to the different groups as shown in Table 20.

On day 23, seven days after the second boost, intratumoral (i.t.) application of 50 μg naked H1N1(PR8)-NP(GC) mRNA (R2650) started (only group C) and continued for additional four injections (at day 25, day 28, day 31 and day 35). The volume for intratumoral injection was 50 μl. A detailed treatment schedule is shown in Table 21.

Tumor growth was monitored by measuring the tumor size in three dimensions using a calliper. Tumor volume was calculated according to the following formula:

${{volume}\mspace{14mu}\left( {mm}^{3} \right)} = \frac{{length}\mspace{14mu}({mm}) \times \pi \times {{width}^{2}\left( {mm}^{2} \right)}}{6}$

TABLE 20 Animal groups Strain Number mRNA Group sex of mice i.d. mRNA i.t. A BALB/c 9 RiLa — Female B BALB/c 9 R2651 — Female (40 μg) C BALB/c 9 R2651 R2650 Female (40 μg) (50 μg)

TABLE 21 Vaccination schedule Day Treatment 0 i.d. vaccination all groups 7 i.d. vaccination all groups 14 Tumor challenge of all groups (1 × 10⁶ CT26 cells/mouse) 16 i.d. vaccination all groups 23 i.t. vaccination group C 25,28, 31,35 i.t. vaccination group C

Results of Example 3

FIG. 4 shows that pre-existent immunity (induced in this model by the NP vaccination) increased the median survival time (MST) of mice which received intratumoral application of NP-encoded mRNA compared to mice which were treated with buffer only (MST=28 vs. MST=21, respectively).

Example 4: Intratumoral Treatment with an Immunostimulating RNA (“RNAdiuvant”) and an mRNA Encoding Soluble PD-1 and an mRNA Encoding IL-12

Table 22 summarizes the treatment as used in the present example. RNAdjuvant and the mRNA constructs encoding IL-12 and soluble PD-1 were administered intratumorally (i.t.). In CT26 tumor challenged mice, survival rates and median tumor growth were analyzed.

TABLE 22 Groups, treatment and RNA dilution Nr. of i.t treatment Vaccination Group mice (25 μg for each component schedule A 10 IL-12 + RNAdjuvant + soluble 2× week PD-1 B 10 IL-12 2× week C 10 RNAdjuvant 2× week D 10 RiLa 2× week

Tumor Challenge and Administration of the Inventive Composition:

60 Balb/c mice were challenged subcutaneously with 1×10⁶ CT26 cells per mouse (volume in 100 μl PBS) on the right flank on day 0 of the experiment. On day 8 mice were sorted according to tumor size. According to tumor size, the first vaccination took place on day 8 or 9 (tumors should have a size of about 40-50 mm³). Mice were vaccinated with different combinations of mRNAs and RNAdjuvant according to the table above. Six vaccinations took place. Volume for intratumoral injection was 50 μl.

Mice were injected according to the indicated scheme shown in Table 22. Median tumor growth was determined according to example 3. The results of the experiment are shown in FIG. 5, wherein FIG. 5A shows the effect of the inventive composition on tumor growth, and FIG. 5B shows the effect of the inventive composition on survival.

Results:

The results in FIG. 5A show that the inventive composition comprising an mRNA encoding IL-12 and mRNA encoding soluble PD-1 in combination with RNAdjuvant (group “A” according to Table 22) to strongly decreased the median tumor volume compared to the other treatments (groups B-D according to Table 22). In addition, the results in FIG. 5B show that the inventive composition comprising an mRNA encoding IL-12 and mRNA encoding soluble PD-1 in combination with RNAdjuvant (group “A” according to Table 22) strongly increased the survival of tumor challenged mice compared to the other treatments (groups B-D according to Table 22).

Example 5: Intratumoral Treatment with mRNA Encoding IL-12 in Combination with an Immunostimulating RNA (“RNAdjuvant”) and mRNA Encoding Sol PD-1 and Anti-CD73 Antibody

Table 23 summarizes the treatment as used in the present example. In addition to RNAdjuvant and mRNA constructs encoding IL-12 and soluble PD-1 (administered intratumorally (i.t.)), an anti CD73 antibody (BioXCell) was co-administered intraperitoreally (i.p.). In CT26 tumor challenged mice, survival rates were analyzed.

TABLE 23 Groups, treatment and RNA dilution Nr. of i.t. treatment i.p. Vaccination Group mice (25 μg for each component) treatment schedule A 10 IL-12 + RNAdjuvant + soluble a-CD73 2× week PD-1 B 10 IL-12 + RNAdjuvant + soluble Rat IgG2a 2× week PD-1 C 10 RiLa a-CD73 2× week D 10 RiLa Rat IgG2a 2× week

Tumor Challenge and Administration of the Inventive Composition:

The tumor challenge was performed according to the previous experiments (see Example 4).

Mice were injected according to the indicated scheme shown in Table 23.

The results of the experiment are shown in FIG. 6.

Results:

FIG. 6 shows that the intratumoral treatment with mRNA-encoded IL-12 (R2763), mRNA encoded sol-PD-1 (R3971) and RNAdjuvant® (R2391) in combination with an i.p. administration of anti CD73 antibody (Group “A” according to Table 23) resulted in a statistically significant increase in survival compared to the relevant control group that only received an anti CD73 antibody (Group “C” according to Table 23) and in an increase in survival rates compared to the treatment with IL-12+RNAdjuvant+soluble PD-1 and a control antibody (Rat IgG2a, BioXCell) (Group “B” according to Table 23).

Example 6: Intratumoral Treatment with mRNA Encoding IL-12 in Combination with an Immunostimulating RNA (“RNAdjuvant”) and an Anti-CD137 Antibody

Table 24 summarizes the treatment as used in the present example. In addition to RNAdjuvant and the mRNA constructs encoding IL-12 and soluble PD-1 (administered intratumorally (i.t.)), an anti CD137 antibody (BioXCell) was co-administered intraperitoreally (i.p.). In CT26 tumor challenged mice, survival rates were analyzed.

TABLE 24 Groups, treatment and RNA dilution Nr. of i.p. Vaccination Group mice i.t. treatment (25 μg) treatment schedule A 10 IL-12 + RNAdjuvant + soluble a-CD137 2× week PD-1 B 10 IL-12 + RNAdjuvant + soluble Rat IgG2a 2× week PD-1 C 10 RiLa a-CD137 2× week D 10 RiLa Rat IgG2a 2× week

Tumor Challenge and Administration of the Inventive Composition:

The tumor challenge was performed according to the previous experiments (see Example 4).

Mice were injected according to the indicated scheme shown in Table 24.

The results of the experiment are shown in FIG. 7.

Results:

FIG. 7 shows that the intratumoral treatment with mRNA-encoded IL-12 (R2763) sol-PD-1 (R3971) and RNAdjuvant® (R2391) in combination with an i.p. administration of anti CD-137 antibody (Group “A” according to Table 24) resulted in a significant increase in survival compared to the relevant control group that only received the antibody anti CD-137 (Group “C” according to Table 24) and in an increase in survival rates compared to the treatment with IL-12+RNAdjuvant+soluble PD-1 and a control antibody (Rat IgG2a, BioXCell) (Group “B” according to Table 24).

Example 7: Treatment with an Immunostimulating RNA (“RNAdjuvant”) in Combination with a Checkpoint Inhibitor Anti PD-1 Antibody

Table 25 summarizes the treatment as used in the present example. In addition to RNAdjuvant (administered i.t.), a checkpoint inhibitor anti PD-1 (BioXCell) was administered i.p. In CT26 tumor challenged mice, survival rates were analyzed.

TABLE 25 Groups, treatment and RNA dilution /antibody dilution Amount of Vaccination Group Construct Antibody RNA (μg) schedule A RiLa (i.t.) — 2× week B RNAdjuvant Control Ab (i.p.)(100 μg) 25 2× week (i.t.) C RNAdjuvant Anti-PD-1 (i.p.) (200 μg) 25 2× week (i.t.) D RiLa (i.t.) Anti-PD-1 (i.p.) (200 μg) — 2× week

Tumor Challenge and Administration of the Inventive Composition:

The tumor challenge was performed according to the previous experiments (see Example 4).

Mice were injected according to the indicated scheme shown in Table 25.

The results of the experiment are shown in FIG. 8.

Results:

FIG. 8 shows that the intratumoral (i.t.) treatment with RNAdjuvant® (R2391) in combination with an i.p. administration of anti PD-1 antibody (Group “C” according to Table 25) resulted in an increase in survival compared to the relevant control group that only received the checkpoint inhibitor anti PD-1 antibody (Group “D” according to Table 25) and in an increase in survival rates compared to the treatment with RNAdjuvant and a control antibody (anti hamster IgG, BioXCell) (Group “B” according to Table 25).

Example 8: Intratumoral Treatment with an Immunostimulating RNA (“RNAdjuvant”) and an mRNA Encoding CD40 Ligand (CD40L) and an mRNA Encoding IL-12

Table 26 summarizes the treatment as used in the present example. RNAdjuvant and the mRNA constructs encoding IL-12 and murine CD40L were administered intratumorally (i.t.). In CT26 tumor challenged mice, survival rates were analyzed.

TABLE 26 Groups, treatment and RNA dilution Nr. of Vaccination Group mice i.t. treatment (25 μg per RNA) schedule A 8 IL-12 + RNAdjuvant + CD40L 2× week B 8 IL-12 2× week C 8 RiLa 2× week

Tumor Challenge and Administration of the Inventive Composition:

The tumor challenge was performed according to the previous experiments (see Example 4).

Mice were injected according to the indicated scheme shown in Table 26.

The results of the experiment are shown in FIG. 9.

Results:

The results in FIG. 9 show that the inventive composition comprising an mRNA encoding IL-12 and an mRNA encoding CD40L in combination with RNAdjuvant (group “A” according to table 26) strongly increased the median survival of tumor challenged mice compared to the other treatments (groups B-C according to table 26). 

The invention claimed is:
 1. A method for treating a subject having cancer comprising administering (a) a composition comprising non-coding immunostimulating RNA (isRNA) of 5 to 1,000 bases in length to the subject by intratumoral injection, wherein the immunostimulating RNA is complexed with one or more cationic or polycationic compounds, and (b) an immune checkpoint modulator selected from a PD-1 inhibitor and a PD-L1 inhibitor.
 2. The method of claim 1, wherein the immunostimulating RNA comprises a sequence at least 90% identical to SEQ ID NO: 5, 394 or
 10072. 3. The method of claim 1, wherein the one or more cationic or polycationic compounds comprise polymers, peptides, polysaccharides, and/or lipids.
 4. The method of claim 1, wherein the cationic or polycationic compound is a polymeric carrier.
 5. The method of claim 4, wherein the polymeric carrier comprises disulfide-crosslinked cationic peptides.
 6. The method of claim 4, wherein the polymeric carrier comprises a cationic peptide CR₁₂C and/or CR₁₂.
 7. The method of claim 1, wherein the immunostimulating RNA comprises a sequence at least 90% identical to SEQ ID NO: 5 and the polymeric carrier comprises a cationic peptides comprising the sequence CR₁₂C, said cationic peptides comprising crosslinks by C-C disulfide bonds.
 8. The method of claim 1, wherein the composition further comprises a mRNA encoding a cytokine.
 9. The method of claim 8, wherein the mRNA encoding a cytokine comprises a 5′-UTR element, a 3′-UTR element, a histone stem-loop, a 5′-CAP structure, a poly(A) sequence, and/or a poly(C) sequence.
 10. The method of claim 1, further comprising administering a cytokine.
 11. The method of claim 10, wherein the cytokine comprises IL-12.
 12. The method of claim 10, wherein the cytokine comprises a mRNA encoding IL-12.
 13. The method of claim 12, wherein the mRNA encoding IL-12 is administered via intratumoral injection.
 14. The method of claim 1, wherein the immune checkpoint modulator is a PD-L1 inhibitor.
 15. The method of claim 1, further comprising administering CD40L or an mRNA encoding CD40L to the subject.
 16. The method of claim 14, wherein the immune checkpoint modulator is an antibody directed against PD-L1. 